Methods and compositions for polytopic vaccination

ABSTRACT

The present invention relates to therapeutic and prophylactic methods for treating or preventing an infectious disease in a subject by stimulating or enhancing an immune response against an infectious agent causing the disease. The methods comprise administering to the subject a plurality of compositions, each composition being administered to a different site of the subject, wherein each site is, or substantially drains to, an anatomically distinct lymph node, a group of lymph nodes, a nonencapsulated cluster of lymphoid tissue, or the spleen. Each composition comprises at least one antigenic molecule having one or more epitopes of the same infectious agent or a strain thereof. The antigenic molecules of each composition comprise in aggregate a set of epitopes distinct from that of any other composition that is administered to the subject.

The invention described herein was made with government support undergrant number CTS-0243520 from the National Science Foundation.Accordingly, the United States government has certain rights in thisinvention.

1. FIELD OF THE INVENTION

The present invention relates to methods and compositions fortherapeutic and prophylactic vaccination. The methods of the inventionovercome the deleterious effects of immunodominance and provide moreeffective immunity against infectious agents, particularly multi-straininfectious agents.

2. BACKGROUND OF THE INVENTION

A significant problem in vaccine development is overcoming the effectsof the poorly understood phenomenon of immunodominance. Although aparticular infectious agent comprises hundreds or thousands ofpotentially antigenic molecules, each comprising multiple protein orpeptide epitopes capable of binding to antibodies or to MHC molecules,the immune response elicited against a particular infectious agent, isoften directed against only a limited number of epitopes, or even to asingle epitope (see van der Most et al., J. Immunol. 1996 157:5543-54and references infra). These epitopes are referred to as“immunodominant” epitopes. Such a narrow immune response to a fewimmunodominant epitopes offers poor protection against subsequentinfection by a mutated form or by different strains of the originalinfectious agent. The problem of immunodominance is especially acute forinfectious agents having a high mutation rate and for those comprisingmultiple strains.

For reasons that are not well understood, the immune response elicitedby some immunodominant epitopes actually impairs the ability to developan effective response against a subsequent infection. This has beenobserved, for example, with infectious diseases caused by multi-strainpathogens. This phenomenon, also referred to as “original antigenicsin,” was first characterized with respect to the influenza virus(Fazekas de St. Groth and Webster, 1966 J. Exp. Med. 124:331-45), andhas since been observed in hepatitis B and C (Harcourt et al., 2003Clin. Exp. Immunol. 131:122-29), malaria (Good et al., 1993 ParasiteImmunol. 15:187-93), dengue fever (Rothman et al., 2001 Vaccine 19,4694-4699), Chlamydia (Berry et al., 1999 J. Infec. Dis. 179:180-86),and HIV (Anderson et al., 2001 Clin. Immunol. 101:152-57). For example,immunity after infection by a particular strain of dengue virus protectsonly modestly or even negatively against reinfection by one of the otherthree strains (Mongkolsapaya et al. 2003 Nature Medicine 9:921-27). Thiseffect has also been observed for subsequent infections by a differentpathogen. For example, exposure to influenza appears to increasesusceptibility to hepatitis C through immunodominance (Brehm et al. 2002NI 3:627-34). The existence of this phenomenon means that for someinfectious diseases, vaccinated individuals may paradoxically be moresusceptible to infection by another strain of the same pathogen, or evento another pathogen, than individuals who were not vaccinated. An effectsimilar to original antigenic sin has also been observed in the contextof tumor immunity (Makki et al. 2002 Cancer Immunity 2:4-17, andreferences infra; see also Cole et al. 1997 J. Immunol. 158: 4301-09 andvan der Most et al. 1996 J. Immunol. 157:554354). Makki et al. suggestthat vaccination with certain dominant tumor antigens not only fails toelicit an immune response against the tumor, but also hinders thedevelopment of an effective response against other, presumablysubdominant tumor antigens.

Immunodominance has also been observed in the context of tumor immunity.For example, an existent response to a tumor antigen may prevent aresponse to new tumor antigens arising through mutation. Schreiber etal. refers to this phenomenon as the “priority of the first response”which was suggested by experiments in mice showing that repeatedimmunization with an antigen A, followed by later immunization with anantigen B, fails to elicit an anti-B response (Schreiber et al. 2002Cancer Biol. 12:25-31, and references infra). This immunodominance couldbe broken experimentally by vaccination with individual tumor antigensat separate sites, rather than with multiple antigens at one site.

One factor in determining whether an epitope becomes dominant appears tobe its effectiveness in generating an immune response (Schreiber et al.,Cancer Biol. 2002 12:25-31). This is a function of a number of factors,including the binding affinity of the epitope for T cell receptors orfor antibodies expressed by B cells. Intracellular processing of peptideantigens is also a factor because epitopes which are presented at highlevels on the surface of antigen presenting cells tend to elicit astronger response. However, it is not simply the case that the dominantepitopes are able to elicit an immune response, and subdominant epitopesare not. The ability of subdominant epitopes to elicit an immuneresponse has been demonstrated, for example, in the context of viralinfections and tumor immunity (see Cole et al, J. Immunol. 1997158:4301-09; van der Most et al., J. Immunol. 1996 157:5543-54; Makki etal., Cancer Immunity 2002 2:4-17 and references infra). Makki et al.suggests that, in the context of tumor immunity, vaccination with asubdominant epitope may even be superior to vaccination with a dominantepitope.

It is not known why subdominant epitopes which are capable of elicitingan immune response nevertheless often fail to do so. However, there isevidence that dominant epitopes can suppresses immunity to the other,subdominant epitopes. This phenomenon may be a result of the manner inwhich antigen-specific effector cells are selected. For example,cytotoxic T cells (“CTLs” or “CD8+ T cells”) binding to MHC-peptidecomplexes on an antigen presenting cell can inhibit the proliferation ofother CTLs binding to other complexes on the same cell. Since suchbinding is required to stimulate T cell proliferation, and onlyproliferating T cells mature into memory T cells, the effect ispresumably to produce narrowing of the repertoire of memory T cells.Thus, the ability to protect against a secondary infection by a similarbut not identical infectious agent is reduced. This and other aspects ofthe cellular biology and immunology of immuodominance in the cytotoxic Tcell response against viral infections are reviewed by Yewdell and DelVal, Immunity 2004 2:149-53.

There remain fundamental unanswered questions that have hindered thedesign of effective vaccines or vaccination strategies that willeffectively avoid the adverse effects of immunodominance. For example,the relationship between antibody or T cell receptor binding, thesequence of the antigen, and the emergence of immunodominance is notknown. Understanding these relationships is important to vaccine designgenerally and is of particular importance to the development of safe,effective peptide-based vaccines. For a review of epitopeidentification, vaccine design and delivery, see Sette and Fikes, 2003Cur. Opinion Immunol. 15:461-70.

One approach to answering these questions is to utilize mathematicalmodels that capture the sequence-level dynamics of the effector cell andepitope binding interactions. A random energy model is one suchmathematical model which has successfully reproduced complex immunephenomena such as immunodominance and original antigenic sin. This modelcaptures much of the thermodynamics of protein folding and ligandbinding, and consequently also captures the correlations between thethree dimensional amino acid structure of antibodies or T cell receptorsand the amino acid sequences of antigenic molecules. The specificantibody or T cell repertoire of an individual is represented in themodel by a specific set of amino acid sequences. An epitope of aspecific antigen or viral strain is represented by a specific instanceof the random parameters. An immune response that finds a T cellreceptor or antibody with a high binding affinity to a specific epitopecorresponds in the model to finding an amino acid sequence having a lowenergy for a specific parameter set.

The robustness of the model as a tool for accurately simulating theinteractions between effector cells and antigens has been demonstratedby a number of experiments. For example, Deem and Lee demonstrated thatoriginal antigenic sin in the context of influenza stems from thelocalization of the immune system response in antibody sequence space(Phys. Rev. Lett. 2003 91:68101-104). This localization stems frommemory sequences being less able to evolve than naïve sequences, and isobserved in general for diseases with high year-to-year mutation rates,such as influenza. Building on these results, Deem and Munozdemonstrated that this localization played a role in the ineffectivenessof the 2003-2004 influenza vaccine in the United States (Vaccine 200523:1144-48). Predictions from the model also correlated well with theefficacies of the H3N2 influenza A component of the annual vaccinebetween 1971 and 2004 (Gupta and Deem 2005 Quant. Biol., document no.0503030). In fact, the predictive value of the model was superior tothat of the standard ferret animal model.

In another example, the model was used to examine cross-reactivity inthe T cell response to mutated viral antigens (Park and Deem 2004Physica A. 341:455-70). Here again, the predicted specific lysis curveswere in excellent agreement with ex vivo and in vitro altered peptideligand experiments. Predictions from the model of immunodominance in thehuman immune response to the four-component dengue vaccine alsoaccurately predicted experimental results (Deem 2004 AIChE J.50:734-38).

It is clear from these results that the model is able to accuratelysimulate important aspects of the immune response to an antigen andthereby provide insights for vaccine design and development. The presentinvention is based in part on such an insight from the model, namelythat multi-site vaccination against an infectious agent increasesimmunity against subdominant epitopes, thereby mitigating the effects ofimmunodominance.

3. SUMMARY OF THE INVENTION

The invention provides a method of treating or preventing an infectiousdisease in a subject comprising administering to the subject a pluralityof compositions, each composition being administered to a different siteof the subject, wherein each composition comprises at least oneantigenic molecule, wherein at least one antigenic molecule in eachcomposition comprises one or more epitopes of the same infectious agentor a strain thereof, and wherein the one or more molecules of eachcomposition comprise in aggregate a set of epitopes distinct from thatof any other composition so administered, wherein each site is, orsubstantially drains to, an anatomically distinct bodily part selectedfrom the group consisting of a lymph node, a group of lymph nodes, anonencapsulated cluster of lymphoid tissue, and the spleen, and whereinthe infectious agent causes the infectious disease.

The invention also provides a method of treating or preventing aninfectious disease in a subject comprising administering to the subjecta plurality of compositions, each composition being administered to adifferent site of the subject wherein each composition comprises atleast one antigenic molecule, wherein at least one antigenic molecule ineach composition comprises one or more epitopes of the same infectiousagent or a strain thereof, and wherein the one or more molecules of eachcomposition comprise in aggregate a set of epitopes distinct from thatof any other composition so administered, wherein the distance betweeneach pair of sites is greater than the distance between any twoanatomically distinct lymph nodes or groups of lymph nodes nearest eachsite, and wherein the infectious agent causes the infectious disease.

The invention also provides a method of treating or preventing one ormore infectious diseases in a subject comprising administering to thesubject a plurality of compositions, each composition being administeredto a different site of the subject wherein each composition comprises atleast one purified antigenic molecule, wherein at least one purifiedantigenic molecule in each composition comprises one or more epitopes ofone or more infectious agents or strains thereof, the epitopes having anepitopic variance of between 0.05 and 0.50, and wherein the one or morepurified antigenic molecules of each composition comprise in aggregate aset of epitopes distinct from that of any other composition soadministered, wherein the distance between each pair of sites is greaterthan the distance between any two anatomically distinct lymph nodes orgroups of lymph nodes nearest each site, wherein said one or moreinfectious agents cause the one or more infectious diseases.

The invention further provides a method for determining whether avaccine composition suppresses immunodominance in a subject comprising(a) administering to the subject a plurality of compositions, eachcomposition being administered to a different site of the subjectwherein each composition comprises at least one antigenic molecule,wherein at least one antigenic molecule in each composition comprisesone or more epitopes of the same infectious agent or a strain thereof,and wherein the one or more molecules of each composition comprise inaggregate a set of epitopes distinct from that of any other compositionso administered, wherein the distance between each site is greater thanthe distance between any two anatomically distinct lymph nodes or groupsof lymph nodes nearest each site; and (b) measuring an immune responseto the at least one antigenic molecule of each composition soadministered, wherein immunodominance has been suppressed in the subjectif the ratio between the immune response of the most immunogeniccomposition and the least immunogenic composition is reduced compared tothe ratio obtained with administration of all of the compositions at asingle site.

The invention also provides a kit comprising in separate containers atleast two compositions, each composition comprising at least oneantigenic molecule and each antigenic molecule comprising one or moreepitopes of the same infectious agent or a strain thereof, wherein theone or more molecules of each composition comprise in aggregate a set ofepitopes distinct from that of said other composition or compositions,and instructions for administering each composition to a separate siteof a subject.

The invention also provides a kit comprising in separate containers atleast two compositions, each composition comprising at least onepurified antigenic molecule, and each purified antigenic moleculecomprising one or more epitopes of one or more infectious agents orstrains thereof, the epitopes having an epitopic variance of between0.05 and 0.50, wherein the one or more purified antigenic molecules ofeach composition comprise in aggregate a set of epitopes distinct fromthat of said other composition or compositions, and instructions foradministering each composition to a separate site of a subject.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Specific lysis ratios for the least to the most dominant epitopeof the four strains of dengue fever. The error bars are given bystandard error analysis of the experimental error bars.

FIG. 2( a)-(c): (a) Evolved clearance probability to a second strainafter exposure to an original strain that differs in the epitope aminoacid sequence by fraction p_(epitope) (solid line). The primary responsein the absence of a vaccine is shown (dashed line, independent ofp_(epitope)). For dengue, p_(epitope)=0.5/9 (circle). (b) During asecondary response for dengue, memory T cell receptors are initiallybetter but eventually worse after selection than are naïve T cellreceptors (p_(epitope)=0.5/9). This result explains the cause oforiginal antigenic sin. (c) The immunodominance among the four strainsunder different conditions. Left column: four strains independentlyevolve during a primary response, and the immunodominance due to theheterologous nature of immunity is measured. That is, the response tostrain i is measured after a primary vaccination against strain i.Middle columns: the i^(th) column shows the response to the four strainsafter a primary vaccination against strain i. Note the immunodominance.That is, the response to strain j is measured after a primaryvaccination against strain i. Right columns: initial primary response tostrain i for the i^(th) column, followed by a secondary response as inthe left column. That is, the response to strain j is measured after aprimary vaccination against strain i and a secondary vaccination againststrain j. For each column, the strains are ranked in order ofimmunodominance.

FIG. 3: Predictions of the theory for a polytopic four-component denguevaccine, p_(epitope)=0.5/9, when the different strains are injected indifferent physical locations and evoke an immune response that evolvesindependently in different lymph nodes until mixing round after whichthe lymph system is well-mixed.

FIG. 4( a)-(d): p_(epitope)=0.5/9. (a) Immunodominance for afour-component dengue vaccine. (b) Reduced immunodominance for a primarysingle-component dengue vaccine (strain i in the i^(th) column),followed by a secondary four-component dengue vaccine. (c) Reducedimmunodominance for a polytopic four-component dengue vaccine, with eachcomponent administered to a distinct lymph node (mixing round=9). (d)Reduced immunodominance for a primary single-component dengue vaccine(strain i in the i^(th) column), followed by a secondary polytopicfour-component dengue vaccine (mixing round=9). An improved response tothe subdominant strain provides the most immunological benefit in thecase of dengue; this response is improved 101% in d) versus a).

FIG. 5( a)-(d): p_(epitope)=1.0/9. (a) Immunodominance for afour-component dengue vaccine. (b) Reduced immunodominance for a primarysingle-component dengue vaccine (strain i in the i^(th) column),followed by a secondary four-component dengue vaccine. (c) Reducedimmunodominance for a polytopic four-component dengue vaccine, with eachcomponent administered to a distinct lymph node (mixing round=9). (d)Reduced immunodominance for a primary single-component dengue vaccine(strain i in the i^(th) column), followed by a secondary polytopicfour-component dengue vaccine (mixing round=9). An improved response tothe subdominant strain provides the most immunological benefit in thecase of dengue; this response is improved 290% in d) versus a).

FIG. 6( a)-(b): (a) For the highest affinity T cell receptor, thebinding constant fluctuates between 10⁵ l/mol and 10⁷ l/mol. (b) Mostbinding constants differ within a factor of 10 for the 0.5% of theantigen specific T cells selected to be memory T cells.

FIG. 7: Flow diagram of the T cell receptor selection dynamics in theGeneralized NK model.

5. DETAILED DESCRIPTION OF THE INVENTION

This invention provides methods for treating or preventing an infectiousdisease in a subject by eliciting an immune response against aninfectious agent which causes the disease. The terms “elicit,”“stimulate,” and “induce” are used interchangeably to denote thegeneration of a de novo immune response in a subject or to denote theenhancement of the strength or persistence of an existing immuneresponse. An infectious agent may be, for example, a pathogen, such as abacterium, a virus, or a parasite. An infectious agent may be a strainof a pathogen, particularly a strain of a virus. Infectious agents andstrains are described in more detail in Section 5.3.

The methods comprise administering to the subject a plurality ofcompositions, each composition being administered to a different site ofthe subject, preferably a human subject, wherein each compositioncomprises at least one antigenic molecule. In one embodiment, at leastone antigenic molecule in each composition comprises one or moreepitopes of the same infectious agent or a strain thereof and the one ormore molecules of each composition comprise in aggregate a set ofepitopes distinct from that of any other composition administered to thesubject. In another embodiment, at least one antigenic molecule in eachcomposition comprises one or more epitopes of one or more infectiousagents, which can be the same or different infectious agents, or strainsthereof, the epitopes having an epitopic variance of between 0.05 and0.50, and wherein the one or more molecules of each composition comprisein aggregate a set of epitopes distinct from that of any othercomposition so administered. In a specific embodiment, the one or moreantigenic molecules are purified; thus, in such an embodiment, forexample, the antigenic molecules are not contained in whole viralparticles. In one embodiment, the epitopes have an epitopic variance ofbetween 0.05 and 0.25. In one embodiment, each site is, or substantiallydrains to, an anatomically distinct bodily part selected from the groupconsisting of a lymph node, a group of lymph nodes, a nonencapsulatedcluster of lymphoid tissue, and the spleen. In another embodiment, thedistance between each pair of sites is greater than the distance betweenany two anatomically distinct lymph nodes or groups of lymph nodesnearest each site. A more detailed description of the sites and methodsof administration, epitopes and antigenic molecules, infectious agents,and pharmaceutical compositions provided by the invention is set forthin the sections that follow.

In one embodiment, the plurality of compositions comprises 2-10compositions. In a preferred embodiment, the plurality of compositionscomprises three or four compositions. In a preferred embodiment, thecompositions are administered to the subject at substantially the sametime. In one embodiment, the total time between the first administrationand the last administration is 5 or fewer days. The compositions maycomprise dominant or subdominant epitopes. In certain embodiments, atleast one composition is administered prior to the other compositions,for example more than 6 days, more than 14 days, or within 6 monthsbefore the other compositions. In a preferred embodiment, at least onecomposition is administered 6-14 days, 1-3 months, 3-6 months, 6-9months, or 9-12 months prior to the other compositions. Preferably, thecomposition administered early comprises one or more subdominantepitopes.

In one embodiment, each composition comprises at least one antigenicmolecule comprising one or more epitopes of a strain of a virus causingthe infectious disease, and no two compositions comprise epitopes of thesame strain. In one embodiment, each composition comprises at least onepurified antigenic molecule comprising one or more epitopes of one ormore viral strains causing the one or more infectious diseases, and notwo compositions comprise epitopes of the same strains. In oneembodiment, each composition comprises at least one antigenic moleculecomprising one or more epitopes of a strain of a virus causing theinfectious disease, and at least two compositions do not compriseepitopes of the same strain. In one embodiment, each compositioncomprises at least one purified antigenic molecule comprising one ormore epitopes of one or more viral strains causing the one or moreinfectious diseases, and at least two compositions do not compriseepitopes of the same strain. In one embodiment, each compositioncomprises at least one antigenic molecule comprising one or moreepitopes of a strain of a virus causing the infectious disease, and notwo compositions comprise the same epitopes. In one embodiment, eachcomposition comprises at least one purified antigenic moleculecomprising one or more epitopes of one or more viral strains causing theone or more infectious diseases, and no two compositions comprise thesame epitopes. In another embodiment, each composition comprises atleast one antigenic molecule comprising one or more subdominant epitopesof an infectious agent causing the infectious disease. In oneembodiment, each composition comprises at least one purified antigenicmolecule comprising one or more subdominant epitopes of one or moreinfectious agents causing the one or more infectious diseases. In oneembodiment, at least one antigenic molecule comprises one or moredominant epitopes of an infectious agent causing the infectious disease.In one embodiment, each composition comprises at least one purifiedantigenic molecule comprising one or more dominant epitopes of one ormore infectious agents causing the one or more infectious diseases.

In one embodiment, the set of epitopes of each composition differs fromevery other set by an epitopic variance defined as the sum of the numberof non-conservative amino acid changes and one half of the number ofconservative amino acid changes divided by the total number of aminoacids in the epitope, and wherein the epitopic variance between theepitopes of each composition is between 0.02 and 1. In anotherembodiment, the epitopic variance between the set of epitopes of eachcomposition is between 0.05-0.95, between 0.05-0.75, between 0.05-0.50,or between 0.02-0.40. Preferably, the epitopic variance among theepitopes within the same composition is less than 0.10.

In one embodiment, each antigenic molecule is a peptide, the amino acidsequence of which consists of 25 amino acids or less. In one embodiment,each antigenic molecule is a peptide, the amino acid sequence of whichconsists of 12 amino acids or less. In specific embodiments, the peptideconsists of 8-11 amino acids, 10-18 amino acids, or 8-18 amino acids.Preferably, the peptide is a T cell epitope or a B cell epitope, andmost preferably a cytotoxic T cell epitope. In one embodiment, theepitopes are overlapping epitopes for different MHC alleles. In anotherembodiment, the epitopes are epitopes of different MHC alleles.

In one embodiment, each site to which a composition is administered isor drains to a lymph node or group of lymph nodes selected from thegroup consisting of the lymph nodes of the head and neck, the axillarylymph nodes, the tracheobronchial lymph nodes, the parietal lymph nodes,the gastric lymph nodes, the ileocolic lymph nodes, and the inguinal andsubinguinal lymph nodes. In another embodiment, the sites are selectedfrom the group consisting of the right arm, the left arm, the rightthigh, the left thigh, the right shoulder, the left shoulder, the rightbreast, the left breast, the abdomen, the right buttock, and the leftbuttock. In one embodiment, the site is or drains to a nonencapsulatedcluster of lymphoid tissue selected from the group consisting of thetonsils, the adenoids, the appendix, and Peyer's patches. In a specificembodiment, at least one composition is administered to a site thatdrains to the spleen.

In one embodiment, each composition is administered by a routeindependently selected from the group consisting of intradermally,subcutaneously, transdermally, intramuscularly, orally, rectally,vaginally, by inhalation, and a combination thereof. In anotherembodiment, each composition is administered by a route independentlyselected from the group consisting of intradermally, subcutaneously,transdermally, intramuscularly, and a combination thereof. In oneembodiment, at least one composition is injected directly into ananatomically distinct lymph node, lymph node cluster, or nonencapsulatedcluster of lymphoid tissue.

In one embodiment, the methods of the invention further compriseadministering to the subject antigen presenting cells which have beensensitized with at least one antigenic molecule of a composition. In apreferred embodiment, the antigen presenting cells are dendritic cells.In one embodiment, the method further comprises administering to thesubject one or more adjuvants. In one embodiment, at least onecomposition further comprises one or more adjuvants. In one embodiment,the one or more adjuvants is selected from the group consisting of anoil-based adjuvant, a CpG DNA adjuvant, a mineral salt adjuvant, amineral salt gel adjuvant, a particulate adjuvant, a microparticulateadjuvant, a mucosal adjuvant, and a cytokine. Further examples ofadjuvants encompassed by the invention are provided by Section 5.4. Suchadjuvants may either be formulated with the compositions of theinvention or administered separately from the compositions, e.g., priorto, concurrently with, or after the compositions are administered to thesubject.

In one embodiment, the prophylactic or therapeutic methods of theinvention are administered to prevent or treat an infectious diseasecaused by an infectious agent which is a virus, a bacterium, aprotozoan, or a parasite. In one embodiment, the virus is selected fromthe group consisting of a lymphocytic choriomeningitis virus, ahepatitis B virus, an Epstein Barr virus, an influenza virus, and ahuman immunodeficiency virus. In one embodiment, the virus is selectedfrom the group consisting of the Flaviviridae family of viruses. In aspecific embodiment, the flavivirus is selected from the groupconsisting of dengue, Kunjin, Japanese encephalitis, West Nile, andyellow fever virus. In a preferred embodiment, the virus is a denguevirus. Additional non-limiting examples of infectious agents encompassedby the methods of this invention are provided in Section 5.3.

The invention also provides a method for determining whether a vaccinecomposition suppresses immunodominance in a subject comprisingadministering to the subject a plurality of compositions, eachcomposition being administered to a different site of the subject andeach composition comprising at least one antigenic molecule as describedherein, and measuring any immune response to the antigenic molecule ormolecules of each composition so administered. The subject may be ahuman subject or a non-human jawed vertebrate. Immunodominance has beensuppressed in the subject if the ratio between the immune response tothe most immunogenic composition and the least immunogenic compositionis reduced compared to the ratio obtained with administration of all ofthe compositions at a single site. In a preferred embodiment, the ratiois reduced by about 20%, about 50%, about 75%, or about 95%. Any routineassay for measuring an immune response to an antigenic molecule may beused in accordance with this embodiment. Preferably, the assay measuresa cytotoxic T cell response to the antigenic molecules of eachcomposition, for example using a tetramer assay or a chromium releaseassay. Further examples of methods for measuring immunodominance bymeasuring an immune response are provided in Section 5.6.

The invention also provides a kit comprising in separate containers atleast two compositions, each composition comprising at least oneantigenic molecule and each antigenic molecule comprising one or moreepitopes of the same infectious agent or a strain thereof, wherein theone or more molecules of each composition comprise in aggregate a set ofepitopes distinct from that of said other composition or compositions,and instructions for administering each composition to a separate siteof a subject. In one embodiment, each composition comprises at least onepurified antigenic molecule, and each purified antigenic moleculecomprises one or more epitopes of one or more infectious agents orstrains thereof, the epitopes having an epitopic variance of between0.05 and 0.50. In a preferred embodiment, the kit comprises three orfour compositions. In another preferred embodiment, the kit furthercomprises in one or more additional containers an anti-viral agent, ananti-bacterial agent, a cytokine, or an adjuvant. The kits provided bythe invention are described more fully in section 5.4.4.

The following sections provide a further description of various aspectsof the invention including sites of administration, epitopes ofantigenic molecules, infectious agents, pharmaceutical compositions,combination therapy, and methods of measuring and monitoringimmunodominance.

5.1 Sites of Administration

The methods of the present invention encompass administering a pluralityof compositions to separate sites of a subject. In a preferredembodiment, each site is, or substantially drains to, an anatomicallydistinct bodily part selected from a lymph node, a group of lymph nodes,a nonencapsulated cluster of lymphoid tissue, or the spleen. In anotherpreferred embodiment, the distance between each site is greater than thedistance between any two anatomically distinct lymph nodes or groups oflymph nodes nearest each site.

In one embodiment, the sites of administering are selected from thegroup consisting of the head, the neck, the right arm, the left arm, theright thigh, the left thigh, the right buttock, the left buttock, andthe abdomen, and combinations thereof. The sites may also be chosen toadminister one or more compositions to an internal organ whichsubstantially drains to a particular group of lymph nodes, for example,the lungs, the stomach, the intestines, or the rectum.

In one embodiment, one or more sites of administering are selected todrain to one or more of the superficial lymph nodes of the head andneck. The superficial lymph nodes of the head and neck include, forexample, the inferior deep cervical glands, the superior deep cervicalglands, the superficial cervical glands, the submental glands, thesubmaxillary glands, the supramandibular glands, the buccinator glands,the parotid glands, the maxillary glands, the posterior auricularglands, and the occipital glands.

In one embodiment, one or more sites of administering are selected todrain to one or more of the axillary lymph nodes (lymphoglandulaeaxillares). The axillary lymph nodes include, for example, the lateralgroup which drains most of the arm; the anterior or pectoral group whichdrains the skin and muscles of the anterior and lateral thoracic wallsas well as the central and lateral parts of the mamma; the posterior orsubscapular group which drains the skin and muscles of the lower part ofthe back of the neck and of the posterior thoracic wall; the central orintermediate group of three or four large glands which drains to thesubclavicular group; and the medial or subclavicular group which drainsto the upper peripheral part of the mamma, but which receives theefferents of all the other axillary glands. Preferably, if one site ofadministering drains to the medial or subclavicular group, the othersites do not drain to the axillary lymph nodes.

In one embodiment, one or more sites of administering are selected todrain to one or more of the tracheobronchial lymph nodes. These glandsdrain the lungs and bronchi, the thoracic part of the trachea, and theheart.

In one embodiment, one or more sites of administering are selected todrain to one or more of the lymph nodes of the stomach or colon,including for example the gastric glands, the subpyloric glands, thepancreatic glands, and the ileocolic glands.

In one embodiment, one or more sites of administering are selected todrain to one or more of the superficial glands of the lower extremity,for example, the inguinal and the subinguinal glands.

In one embodiment, one or more sites of administering are selected todrain to a nonencapsulated cluster of lymphoid tissue, for example, thetonsils and adenoids, the appendix, or Peyer's patches.

The methods of the invention provide for the administration of aplurality of compositions, each to a different site. In a preferredembodiment, the number of compositions administered is 2, 3, 4, or 5.Further examples are provided in Section 5.4.1., Methods ofAdministration.

In a specific example, the method comprises administering twocompositions, each composition being administered to a different site,wherein the first site is selected to drain to one or more of theaxillary lymph nodes and the second site is selected to drain to one ormore of the inguinal lymph nodes (inguinal or subinguinal).

In another specific example, the method comprises administering threecompositions, each composition being administered to a different site,wherein the first site is selected to drain to one or more of theaxillary lymph nodes, the second site is selected to drain to one ormore of the inguinal lymph nodes (inguinal or subinguinal), and thethird site is selected to drain to one or more of the superficial lymphnodes of the head and neck. In another embodiment, the three sites ofadministering are selected to drain to one or more lymph nodes selectedfrom the group consisting of the axillary lymph nodes of the left arm,the axillary lymph nodes of the right arm, the inguinal lymph nodes ofthe left leg, the inguinal lymph nodes of the right leg, thetracheobronchial lymph nodes, and the superficial lymph nodes of thehead and neck.

In another specific example, the method comprises administering fourcompositions, each composition being administered to a different site,wherein the first site is selected to drain to one or more of theaxillary lymph nodes, the second site is selected to drain to one ormore of the superficial lymph nodes of the head and neck, the third siteis selected to drain to one or more of the tracheobronchial lymph nodes,and the fourth site is selected to drain to one or more of thesuperficial glands of the lower extremity, for example, the inguinal andthe subinguinal glands. In another embodiment, the four sites ofadministering are selected to drain to one or more lymph nodes selectedfrom the group consisting of the axillary lymph nodes of the left arm,the axillary lymph nodes of the right arm, the inguinal lymph nodes ofthe left leg, the inguinal lymph nodes of the right leg, thetracheobronchial lymph nodes, and the superficial lymph nodes of thehead and neck.

In a further specific example the method comprises administering fivecompositions, each composition being administered to a different site,wherein the first site is selected to drain to one or more of theaxillary lymph nodes, the second site is selected to drain to one ormore of the superficial lymph nodes of the head and neck, the third siteis selected to drain to one or more of the tracheobronchial lymph nodes,the fourth site is selected to drain to one or more of the superficialglands of the lower extremity, for example, the inguinal and thesubinguinal glands, and the fifth site is selected to drain to one ormore gastric glands. In another embodiment, the five sites ofadministering are selected to drain to one or more lymph nodes selectedfrom the group consisting of the axillary lymph nodes of the left arm,the axillary lymph nodes of the right arm, the inguinal lymph nodes ofthe left leg, the inguinal lymph nodes of the right leg, thetracheobronchial lymph nodes, the superficial lymph nodes of the headand neck, the gastric glands, the subpyloric glands, and the pancreaticglands.

5.2 Antigenic Molecules and Epitopes

The methods of the invention are useful for treating or preventing aninfectious disease in a subject by eliciting an immune response againstan infectious agent which causes the disease. The methods compriseadministering to the subject a plurality of compositions, eachcomposition comprising at least one antigenic molecule. In oneembodiment, each antigenic molecule of a composition comprises one ormore epitopes of the same infectious agent or a strain thereof. Inanother embodiment, each antigenic molecule of a composition comprisesone or more epitopes of one or more infectious agents, which can be thesame or a different infectious agent, or strains thereof, the epitopeshaving an epitopic variance of between 0.05 and 0.50. In a specificembodiment, the one or more antigenic molecules of the composition arepurified; thus, in such an embodiment, for example, the antigenicmolecules are not contained in whole viral particles. The plurality ofcompositions to be administered is selected so that the one or moreantigenic molecules of each composition comprise in aggregate a distinctset of epitopes. Thus, according to the methods of the invention, no twocompositions comprise the same set of epitopes.

As used herein, an epitope is a portion of an antigenic molecule capableof eliciting an immune response to the molecule, preferably a cytotoxicT cell response or an antibody-secreting B cell mediated response, orwhich can be bound by an antibody. The terms “epitope” and “antigenicdeterminant” are used interchangeably herein. The term “antigenic” inthe context of a molecule refers to the ability of the molecule toelicit, stimulate, or induce an immune response to itself, or to bebound by an antibody. Antigenic molecules are usually proteinaceousmolecules such as proteins or polypeptides. The terms “peptide,”“polypeptide,” and “protein” are used interchangeably herein. However,antigenic molecules may also comprise or consist of other molecules suchas carbohydrates, lipids, nucleic acids, or small organic molecules.Small organic molecules preferably comprise one or more cyclic ringstructures. Antigenic molecules may also comprise one or more of theforegoing kinds of molecules, for example, an antigenic molecule can bea glycoprotein, a lipoprotein, a lipopolysaccharide, or a ribonucleicacid.

Preferably, the one or more antigenic molecules of the compositionsadministered according to the methods of the invention are purified fromcontaminating chemical precursors, if chemically synthesized, orsubstantially free of cellular material from the cell or tissue sourcefrom which they are derived. In a specific embodiment, the antigenicmolecules are 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%free of contaminating chemical precursors, proteins, lipids or nucleicacids. In a preferred embodiment, the one or more antigenic molecules ofa composition are substantially free of contaminating virus. Preferably,each composition for administering to a subject is at least 95%, atleast 97%, or at least 99% free of contaminating virus.

In one embodiment, an antigenic molecule of a composition is a peptidehaving an amino acid sequence in the range of 6 to 200 amino acids, 6 to90 amino acids, 6 to 80 amino acids, 6 to 70 amino acids, 6 to 67 aminoacids, 6 to 65 amino acids, 6 to 60 amino acids, 6 to 57 amino acids, 6to 55 amino acids, 6 to 50 amino acids, 6 to 47 amino acids, 6 to 45amino acids, 6 to 40 amino acids, 6 to 37 amino acids, 6 to 35 aminoacids, 6 to 30 amino acids, 6 to 27 amino acids, 6 to 25 amino acids, 6to 20 amino acids, 6 to 17 amino acids, 6 to 15 amino acids, or 6 to 10amino acids.

In another embodiment, an antigenic molecule of a composition is aprotein having an amino acid sequence in the range of 200 to 2000 aminoacids, or 300 to 3000 amino acids. In specific embodiments, theantigenic molecule is a protein having an amino acid sequence in therange of 200 to 1500 amino acids, 200 to 1000 amino acids, 200 to 500amino acids, 300 to 2500 amino acids, 300 to 2000 amino acids, 300 to1500 amino acids, 300 to 1000 amino acids, or 300 to 500 amino acids. Ina preferred embodiment, the antigenic molecule is a protein having anamino acid sequence in the range of 200 to 500, 500 to 1000, 1000 to2000, or 2000 to 3000 amino acids.

In one embodiment, an epitope of the antigenic molecule is a peptide ofabout 6 to 9 amino acids, 6 to 12 amino acids, 6 to 14 amino acids, 6 to16 amino acids, 6 to 18 amino acids, 8 to 11 amino acids, or 10 to 18amino acids. In one embodiment, an epitope of an antigenic molecule is apeptide of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids.

The antigenic molecules of the invention may comprise epitopes that areB cell specific or T cell specific epitopes. For example, the epitopesof the NS3 region of the dengue virus are generally T cell specificepitopes, while those of the structural glycoprotein E may bind toantibodies on the surface of B cells (see Rothman, Adv. Virus Res. 200360:397-419). Specific examples of T-cell epitopes on dengue viralproteins include the following epitopes, amino acid numbering accordingto Rothman and references therein: C (47-55); C (83-92); NS3 (71-79);NS3 (146-154); NS3 (202-211); NS3 (222-231); NS3 (224-234); NS3(235-243); NS3 (241-249); NS3 (255-264); NS3 (351-361); NS3 (500-508);and NS3 (527-535). Examples of immunodominant peptides of anotherflavivirus, the Murray Valley encephalitis virus, are described byRegner et al. in J. Immunol. 2001 166:3820-28. For a description of theantigenic structure of flavivirus proteins generally, see Roehrig, Adv.Virus Res. 59:141-75.

Epitopes capable of eliciting a B cell mediated response include, forexample, peptides that bind with high affinity to an antibody of aresting B cell, thereby stimulating the B cell to proliferate andsecrete the high affinity antibody. For example, the hemagglutininprotein of the influenza virus comprises a number of B cell specificepitopes and the structural glycoprotein E of dengue virus may bind toantibodies secreted by B cells.

The antigenic molecules of the invention may comprise epitopes selectedfrom epitopes known to be subdominant or dominant epitopes. Preferably,at least one epitope of an antigenic molecule is a subdominant epitope.The preferred measure of the immunodominance of an epitope is, followingimmunization of a subject with multiple epitopes, the relative abilityof the epitope, compared to the other epitopes administered to thesubject, to stimulate a cytotoxic T cell response as measured by acytotoxic T cell assay. Examples of such assays are provided in Section5.6. The dominance of an epitope can also be predicted based on itsbinding affinity to antibodies or T cell receptors. Epitopes having highaffinity for antibodies or T cell receptors are expected to be moredominant than epitopes having a comparably low binding affinity for theantibodies or T cell receptors. However, other factors, such asintracellular processing of the peptide epitope, will affect whether andat what level an epitope is presented at the cell surface andconsequently whether it will elicit a significant immune response.Methods for measuring and monitoring immunodominance are provided inSection 5.6.

Immunodominant epitopes and subdominant epitopes that may be used inaccordance with the methods of the invention are known in the art andinclude, for example, those provided by the following references: vander Most et al. describe dominant and subdominant epitopes of chroniclymphocytic choriomeningitis virus in J. Immunol. 1996 157:5543-54. Forexample, NP (118-126) is a dominant epitope and GP (35-43), GP (99-108),and GP (283-291) are subdominant epitopes (amino acid numberingaccording to van der Most and references therein). Subdominant anddominant epitopes of HIV-1 are described by Lichterfeld et al. in Trendsin Immunology 2005 26:166-71; Corbet et al. J. Gen. Virol. 200384:2409-21; Santra et al. J. Immunol. 2002 168:1847-53; and Goulder etal. J. Exp. Med. 1997 185:1423-33. Subdominate epitopes of Epstein-Barrvirus (“EBV”) are described by Micheletti et al. Eur. J. Immunol. 199929:2579-89; and Duraiswamy et al. Can. Res. 2004 64:1483-89. Examples ofdominant epitopes for EBV are provided by Hollsberg in Scand. J.Immunol. 2002 55:189-95. Bertoletti et al. describe dominant andsubdominant epitopes of hepatitis B virus in J. Exp. Med. 1994180:933-43. Examples of epitopes of hepatitis C virus are provided byHimoudi et al. in J. Virology 2002 76:12735-746. Immunodominance hasalso been recognized in association with the influenza virus, see forexample Meijers et al. J. Mol. Biol. 2005 345:1099-1110. Protectionagainst lethal viral infection by vaccination with subdominant peptideswas demonstrated by Oukka et al. in J. Immunol. 1996 157:3039-45. Sendaivirus epitopes are described by Cole et al. in J. Immunol. 1997158:4301-09 and in J. Virology 1995 69:8057-8060. Busch and Pamerdescribe immunodominance in the context of Listeria monocytogenesinfections in J. Immunol. 1998 160:4441-48. Taracha et al. provideexamples of immunodominance in the vaccination of cattle againstTheileria parva in J. Immunology 1995 155:4854-4890.

In a preferred embodiment, the epitope of an antigenic molecule elicitsa T cell mediated immune response, preferably a cytotoxic T cellresponse, in the subject to whom it is administered according to themethods of the invention. Such epitopes are known in the art andinclude, for example, peptides capable of binding to majorhistocompatibility complex (“MHC”) class I or class II molecules.Preferably, the epitopes are MHC class I binding peptides, for example,peptides that bind to MHC molecules produced by the HLA-A, HLA-B, andHLA-C human genes or the H-2, K, D, and L murine genes, or theirequivalents in other species. The art contains numerous examples of MHCbinding peptides and of peptides which are predicted to bind to MHCmolecules because they comprise known MHC binding sequence motifs. Forexample, the Molecular Immunology Foundation associated with HarvardUniversity maintains a publicly accessible database of peptides ofinfectious agents that bind, or are predicted to bind, to MHC moleculesderived from a number of mammalian species.

In certain embodiments, the amino acid sequence variability of theepitopes of each composition administered according to the methods ofthe invention can be described as “epitopic variance” or “p_(epitope)”.For example, for two epitopes the variance is calculated as the sum ofthe number of non-conservative (“NC”) amino acid changes and one half ofthe number of conservative amino acid changes (“C”) divided by the totalnumber of amino acids (“T”) in the epitope:p _(epitope)=(NC+(0.5*C))/T.

For greater than two epitopes, the epitopic variance is defined as theaverage of this quantity over all pairs of epitopes. Conservative andnon-conservative amino acid changes are known in the art. See, forexample, W. R. Taylor, The Classification of Amino Acid Conservation, J.Theor. Biol. 1986 119:205-218, and D. Bordo and P. Argos, Suggestionsfor “Safe” Residue Substitutions in Site-Directed Mutagensis, 1991 J.Mol. Biol. 217:721-729. Generally, a conservative amino acid changerefers to a substitution of one amino acid for another amino acid havingsubstantially similar chemical properties, specifically with referenceto the amino acid side chains. A non-conservative change refers to asubstitution of one amino acid for another amino acid havingsubstantially different chemical properties. Generally, conservativesubstitutions are those recognized in the art as being unlikely toaffect the overall structure or biological function of the polypeptide,while non-conservative changes are recognized as more likely to affectstructure and function.

Non-limiting examples of a conservative amino change includesubstitution of amino acids within the following groups: aliphatic,aromatic, polar, nonpolar, acidic, basic, phosphorylatable hydrophobic,hydrophilic, small nonpolar, small polar, large nonpolar, and largepolar. Non-limiting examples of non-conservative amino acid changesinclude substitutions of amino acids between any of the foregoinggroups.

In one embodiment, a conservative amino acid change is a substitution inwhich the substitution matrix for the pair of residues has a positivevalue. Examples of amino acid substitution matrices are known in theart, for example the BLOSUM50 matrix or the PAM250 matrix (see W. A.Pearson, Rapid and Sensitive Sequence Comparison with FASTP and FASTA,Meth. Enzymology, 1990 183:63-98, ed. R. Doolittle, Academic Press, SanDiego). For further examples of scoring matrices and a comparisonbetween them see M. S. Johnson and J. P. Overington, 1993, A StructuralBasis for Sequence Comparisons: An Evaluation of Scoring Methodologies,J. Mol. Biol. 233:716-738.

In a preferred embodiment, a conservative amino acid change is asubstitution of one amino acid for another amino acid within the samechemical group wherein the groups are selected from neutral and polaramino acids (Ser, Thr, Pro, Ala, Gly, Asn, Gln), negatively charged andpolar amino acids (Asp, Glu), positively charged and polar amino acids(His, Arg, Lys), nonpolar amino acids lacking a ring structure (Met,Ile, Leu, Val), nonpolar amino acids having a ring structure (Phe, Tyr,Trp), and Cysteine.

It is envisioned that the methods of the invention will be usefulregardless of the specific epitopic variances among the epitopes of theantigenic molecules. However, in certain embodiments, the epitopicvariance may be predictive of the relative ability of two or morecompositions to elicit an immune response. In one embodiment, theepitopic variance refers to the variance between a dominant epitope andone or more subdominant epitopes. In one embodiment, the epitopicvariance is a value between zero (“0”) and 1. In one embodiment, theepitopic variance is from 0.01 to 0.10, from 0.01 to 0.20, from 0.01 to0.30, from 0.01 to 0.40, from 0.01 to 0.50, from 0.01 to 0.60, from 0.01to 0.70, from 0.01 to 0.80, or from 0.01 or 0.90. In another embodiment,the epitopic variance is from 0.10 to 0.80, from 0.10 to 0.70, from 0.10to 0.60, from 0.10 to 0.50, or from 0.10 to 0.40. In a preferredembodiment, the epitopic variance is from 0.02 to 0.50, from 0.02 to0.40, from 0.10 to 0.50, or from 0.10 to 0.40.

Specific non-limiting examples of dominant and subdominant epitopes,along with their calculated epitopic variances, are provided in Table 1;additional examples of calculated epitopic variance values are providedin Table 2; and examples of epitopes for common infectious agents areprovided in Table 3.

TABLE 1Examples of Dominant and Subdominant Epitopes and Epitopic VariancesPathogen Dominant Subdominant P_(epitope)(ratio) P_(epitope) Sequence IDReference Adenovirus YLLEMLWRL YLQQNWWTL (1 + 0.5 + 1 + 0.39SEQ ID NO: 1 Cancer Res.  1 + 1)/9 (2004) 64, 1483-89 Dengue TPEGIIPALTPEGIIPSM 1.0/9 0.11 SEQ ID NO: 2 J. Exp. Med.  (1995) 182:853-63 DengueTPEGIIPAL TPEGIIPTL 0.5/9 0.06 SEQ ID NO: 3 J. Exp. Med. (1995) 182:853-63 Epstein Barr virus CLAGLLTMV CLGGLLTMV 0.5/9 0.06SEQ ID NO: 4 Eur. J. Immunol.  (1999) 29:2579-89 Epstein Barr virusGLCTLVAML LLWTLVVLL (1 + 1 + 1 + 0.39 SEQ ID NO: 5 Scand. J. Immunol. 0.5)/9 (2002) 55, 189-195 Epstein Barr virus IVTDFSVIK AVFDRKSDAK (1 +1 + 1 + 0.78 SEQ ID NO: 6 Annu. Rev. Immunol.  1 + 1 + 1 +(1997) 15:405-31 1)/9 = 7/9 Epstein Barr virus QAKWRLQTL FLRGRAYGL (1 +1 + 0.5 + 0.67 SEQ ID NO: 7 Annu. Rev. Immunol.  1 + 1 + 1 +(1997) 15:405-31 0.5) = 6/9 Epstein Barr virus YLAGLLTMV CLGGLLTMV (1 +0.5)/9 0.17 SEQ ID NO: 8 Eur. J. Immunol.  (1999) 29:2579-89Flaviviridae EEHSGNEI REHRKVAI (1 + 1 + 1 + 0.63 SEQ ID NO: 9J. Immunol. (2001), 1 + 1)/8  166:3820-28 Flaviviridae EEHSGNEI TEHSGNEI1/8 0.13 SEQ ID NO: 10 J. Immunol. (2001), 166:3820-28 FlaviviridaeEEHSGNEI AEHTGREI (1 + 0.5 + 0.31 SEQ ID NO: 11 J. Immunol. (2001), 1)/8166:3820-28 Flaviviridae EEHSGNEI EEHDGNEI 1/8 0.13 SEQ ID NO: 12J. Immunol. (2001), 166:3820-28 Hepatitis B-virus FLPSDFFPSV FLPNDFFPSV1/10 0.10 SEQ ID NO: 13 J. Exp. Med. (1994) 933-43 Hepatitis B virusFLPSDFFPSV FLPNDFFPSA 2/10 0.20 SEQ ID NO: 14 J. Exp. Med. (1994) 933-43Hepatitis B virus FLPSDFFPSV FLPVDFFPSV 1/10 0.10 SEQ ID NO: 15J. Exp. Med. (1994) 933-43 Hepatitis B virus FLPSDFFPSV FLPADFFPSV0.5/10 0.05 SEQ ID NO: 16 J. Exp. Med. (1994) 933-43 Hepatitis B virusFLPSDFFPSV FLPADFFPSI (0.5 + 0.5)/10 0.10 SEQ ID NO: 17J. Exp. Med. (1994) 933-43 Hepatitis C virus DLMGYIPLV ILDSFDPLR (1 +1 + 0.5 + 0.56 SEQ ID NO: 18 J. Virology (2002), 0.5 + 1 + 1)/76, 12735-46 9 = 5/9 HIV GLADQLIHL GLADQLIHM 0.5/9 0.06 SEQ ID NO: 19J. General Virology  (2003) 84:2409-21 HIV NVWATHACY NIWATHACV 0.5/90.06 SEQ ID NO: 20 J. General Virology  (2003) 84:2409-21 HIV RLRPGGKKKRLRPGGKKC 1/9 0.11 SEQ ID NO: 21 J. Exp. Med. (1997),  185, 1423-33 HIVSLVKHHMYV SLVKHHMYI 0.5/9 0.06 SEQ ID NO: 22 J. General Virology (2003) 84:2409-21 HIV SLYNTVATL SLFNTVATL 0.5/9 0.06 SEQ ID NO: 23J. Exp. Med. (1997),  185, 1423-33 Influenza A ASNENMETM SSLENFRAYV(0.5 + 1 + 1 + 0.56 SEQ ID NO: 24 J. Mol. Biol. (2005)  1 + 1 + 0.5)/345, 1099-110 9 = 5/9 Influenza A/PR8/34 ASNENMETM ASNENMDAM (0.5 +0.5)/9 0.11 SEQ ID NO: 25 J. Immunol. (1996) 157:3039-45 ListeriaGYKDGNEYI GYLTDNDEI (1 + 1 + 1 + 0.50 SEQ ID NO: 26 J. Immunol. (1998)0.5 + 1)/9 160:4441-48 Listeria KYGVSVQDI IYVGNGQMI (1 + 1 + 1 + 0.67SEQ ID NO: 27 J. Immunol. (1998) 1 + 1 + 1)/9 160:4441-48 SimianCTPYDINQM STPPLVRLV (1 + 1 + 1 + 0.67 SEQ ID NO: 28 J. Immunol. (2002)Immunodeficiency 0.5 + 1 + 1 + 168:1847-53 virus 0.5)/9 = 6/9

TABLE 2Examples of p-epitope values calculated for peptides used in studiesof cytotoxic T cell vaccines.* TYQRTRA SYIPSAE YPHFMPT RPQASGV EEGAIVGLV KI NL YM SDYEGRLI EI ASNENMDAM SGPSNTPPEI SIINFEKL FAPGNYPALTYQRTRALV 0 0.7222 0.8889 0.7778 0.6875 0.8889 0.8333 0.7222 0.81250.8889 SEQ ID. NO: 29 SYIPSAEKI 0.7222 0 0.8889 0.7222 0.8125 0.83330.8333 0.7222 0.5625 0.8889 SEQ ID. NO: 30 YPHFMPTNL 0.8889 0.8889 00.7778 0.75 0.7778 0.8889 0.6111 0.8125 0.7222 SEQ ID. NO: 31 RPQASGVYM0.7778 0.7222 0.7778 0 0.6875 0.8333 0.7778 0.7222 0.875 0.8333SEQ ID. NO: 32 SDYEGRLI 0.6875 0.8125 0.75 0.6875 0 0.875 0.6875 0.750.75 0.8125 SEQ ID. NO: 33 EEGAIVGEI 0.8889 0.8333 0.7778 0.8333 0.875 00.8889 0.6667 0.9375 0.7778 SEQ ID NO: 34 ASNENMDAM 0.8333 0.8333 0.88890.7778 0.6875 0.8889 0 0.7222 0.625 0.6667 SEQ ID. NO: 35 SGPSNTPPEI0.7222 0.7222 0.6111 0.7222 0.75 0.6667 0.7222 0 0.8125 0.5SEQ ID. NO: 36 SIINFEKL 0.8125 0.5625 0.8125 0.875 0.75 0.9375 0.6250.8125 0 0.625 SEQ ID. NO: 37 FAPGNYPAL 0.8889 0.8889 0.7222 0.83330.8125 0.7778 0.6667 0.5 0.625 0 SEQ ID NO: 38 *See Elliott, S.L. etal., 1999 Vaccine 17:2009-2019.

TABLE 3 Examples of epitopes for some infectious agents*. PathogenSequence Sequence ID: Dengue: TPEGIIPAL SEQUENCE ID: 39 Dengue:TPEGIIPSM SEQUENCE ID: 40 Dengue: TPEGIIPTL SEQUENCE ID: 41 Dengue:GTSGSPIIDKK SEQUENCE ID: 42 Dengue: GTSGSPIVDRK SEQUENCE ID: 43 Dengue:GTSGSPIVDKK SEQUENCE ID: 44 Dengue: GTSGSPIADKK SEQUENCE ID: 45 Dengue:GTSGSPIVNRE SEQUENCE ID: 46 Dengue: GTSGSPIINRE SEQUENCE ID: 47 Dengue:GTSGSPIINRK SEQUENCE ID: 48 Dengue: LAPTRVVAAEME SEQUENCE ID: 49 Dengue:LAPTRVVASEMA SEQUENCE ID: 50 Dengue: DSGCVVSWKNKELKC SEQUENCE ID: 51Dengue: DSGVINWKGRELKC SEQUENCE ID: 52 Dengue: DMGCVINWKGKELKCSEQUENCE ID: 53 Dengue: DMGCVVSWSGKELKC SEQUENCE ID: 54 Dengue:GYISTRVEM SEQUENCE ID: 55 Dengue: GYISTRVGM SEQUENCE ID: 56 Kunji:GYISTRVEL SEQUENCE ID: 57 Murray Valley GYIATRVEA SEQUENCE ID: 58Encephalitis: West Nile Virus: GYIATKVEL SEQUENCE ID: 59 JapaneseGYIATKVEL SEQUENCE ID: 60 Encephalitis: Yellow Fever: GWAAHRARASEQUENCE ID: 61 HIV: GLADQLIHL SEQUENCE ID: 62 HIV: GLADQLIHMSEQUENCE ID: 63 HIV: SLVKHHMYV SEQUENCE ID: 64 HIV: SLVKHHMYISEQUENCE ID: 65 HIV: NVWATHACV SEQUENCE ID: 66 HIV: NIWATHACVSEQUENCE ID: 67 HIV: SLYNTVATL SEQUENCE ID: 68 HIV: SLFNTVATLSEQUENCE ID: 69 HIV: RLRPGGKKK SEQUENCE ID: 70 HIV: RLRPGGKKCSEQUENCE ID: 71 HIV-2: TPYDINQML SEQUENCE ID: 72 HIV-1: TPQDLNMMLSEQUENCE ID: 73 HIV-2: TSTVEEQIQW SEQUENCE ID: 74 HIV-1: TSTLQEQIGWSEQUENCE ID: 75 HIV-1: ALTDICTEM SEQUENCE ID: 76 HIV-1: ALVEICTEMSEQUENCE ID: 77 HIV-1: ALTAICEEM SEQUENCE ID: 78 HIV-1: ALIEICSEMSEQUENCE ID: 79 HIV-1: KMIGGIGGFI SEQUENCE ID: 80 HIV-1: KVIVGIGGFISEQUENCE ID: 81 HIV-1: VLVGPTPVNI SEQUENCE ID: 82 HIV-1: VLVGPTPTNVSEQUENCE ID: 83 HIV-1: VLAEAMSQV SEQUENCE ID: 84 HIV-1: VLAEAMSQASEQUENCE ID: 85 HIV-1: VLAEAMSQI SEQUENCE ID: 86 HIV-1: KLTPLCVTLSEQUENCE ID: 87 HIV-1: KLTPLCVPL SEQUENCE ID: 88 Influenza: ASNENMETMSEQUENCE ID: 89 Influenza: SSLENFRAYV SEQUENCE ID: 90 Influenza:ASNENMETM SEQUENCE ID: 91 Influenza: ASNENMDAM SEQUENCE ID: 92Influenza: SFYRNVVWLIKK SEQUENCE ID: 93 Influenza: SFFRNVVWLIKKSEQUENCE ID: 94 Influenza: SFLRNVVWLIKK SEQUENCE ID: 95 Influenza:ASNENMETM SEQUENCE ID: 96 Influenza: SSLENFRAYV SEQUENCE ID: 97Influenza: SSAENFRAYV SEQUENCE ID: 98 Influenza: SSLENFAAYVSEQUENCE ID: 99 Epstein-Barr YLAGLLTMV SEQUENCE ID: 100 Virus: EBVCLAGLLTMV SEQUENCE ID: 101 EBV: CLGGLLTMV SEQUENCE ID: 102 EBV:GLCTLVAML SEQUENCE ID: 103 EBV: LLWTLVVLL SEQUENCE ID: 104 Heptatitis BFLPSDFFPSV SEQUENCE ID: 105 Virus: HBV: FLPNDFFPSV SEQUENCE ID: 106 HBV:FLPNDFFPSA SEQUENCE ID: 107 HBV: FLPVDFFPSV SEQUENCE ID: 108 HBV:FLPADFFPSV SEQUENCE ID: 109 HBV: FLPADFFPSI SEQUENCE ID: 110 *Additionalinformation regarding the epitopes listed in this table can be found inthe following references: Zivny, et al., J. Exp. Med. 182, 853 C863(1995); Mongkolsapaya, Nat. Med., 9(7):921-7 (2003); Zivna, J. Immunol.168:5959-5965 (2002); Huang, J. Med. Virol., 57:1-8 (1999); Spaulding,J. Virol., 73(1): 398-403 (1999); Corbet, J. of General Virology, 84,2409-21 (2003); Goulder, J. Exp. Med., 185, 1423-1433 (1997); Gillespie,Eur. J. Immunol. 35(2005); Singh, J. Immunol. 173, 4387-4393 (2004);McKinney, J. Immunol. 173, 1941-1950 (2004); Meijers, J. Mol. Biol.,345, 1099-1110 (2005); Oukka, J. Imunol. 157:3039-3045 (1996); Hioe, J.Virol., 64(12), 6246-6251 (1990); Turner, Nat. Immunol., 6(4), 382-9(2005); Micheletti, Eur. J. Immunol. 29: 2579-2589 (1999); Hollsberg,Scand. J. Immunol. 55(2): 189-95 (2002); Bertoletti, J. Exp. Med.,180(3), 933-943 (1994); and Bertoletti, Nature, 369(2) 407-410 (1994).

5.3 Infectious Diseases and Agents

The present invention comprises methods of treating or preventing aninfectious disease in a subject by eliciting an immune response againstone or more infectious agents which cause the disease. In oneembodiment, the methods comprise treating or preventing one or moreinfectious diseases caused by one or more infectious agents. Infectiousagents encompassed by the methods of the invention include, withoutlimitation, viruses, bacteria, fungi, protozoa, helminths, andparasites, and particular strains thereof. The methods of the inventionare particularly useful in eliciting an effective immune responseagainst one or more infectious agents having a high rate of mutation orhaving multiple subtypes or “strains.” As used herein, a “strain” refersto a sub-group of a given species of infectious agent that differsslightly in some of its features with respect to the other strains ofthe same infectious agent. The difference may be defined by the DNAsequence, by the response to a biochemical assay, by antigenicity, or bypathology in the host. Several strains of a single species of infectiousagent can coexist in a population of hosts or even in a single host.Further non-limiting examples of infectious agents and strains thereofwhich are encompassed by the methods of the invention are provided inthe sections that follow.

5.3.1 Viruses

In certain embodiments of the invention, the infectious agent is avirus, preferably a multi-strain virus. Non-limiting examples of suchviruses include herpes viruses (HSV-1, HSV-2, VZV, EBV, CMV, HHV-6,HHV-8), influenza viruses (Flu A, B), hepatitis viruses (HepA, HepB,HepC, HepE), human immunodeficiency viruses (HIV-1, HIV-2), respiratorysyncytial viruses, measles viruses, rhinoviruses, adenoviruses, SARSviruses, papillomaviruses, orthopoxviruses, West Nile viruses, and adengue viruses. In one embodiment, the virus is a member of theFlaviviridae family of viruses. In a preferred embodiment, the virus isa flavivirus selected from the group consisting of dengue, Kunjin,Japanese encephalitits, West Nile, and yellow fever virus.

In one embodiment, the virus is one that is known to escape immunesurveillance by mutation of immunodominant T cell epitopes. Non-limitingexamples of such viruses include lymphocytic choriomenignitis virus,hepatitis B virus, Epstein Barr virus, and human immunodeficiency virus.

Other examples of viruses encompassed by the methods of the inventioninclude, without limitation, the following viruses: Retroviridae (e.g.human immunodeficiency viruses, such as HIV-1, also referred to asHTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such asHIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus;enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae(e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g.dengue viruses, encephalitis viruses, yellow fever viruses);Coronaviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicularstomatitis viruses, rabies viruses); Filoviridae (e.g. ebola-likeviruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus,measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic feverviruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses);Bomaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2), varicella zoster virus, cytomegalovirus (CMV), herpesvirus; Poxyiridae (variola viruses, vaccinia viruses, pox viruses); andIridoviridae (e.g. African swine fever virus); and unclassified viruses(e.g. the etiological agents of Spongiform encephalopathies, the agentof delta hepatitis, thought to be a defective satellite of hepatitis Bvirus), the agents of non-A, non-B hepatitis (class 1, internallytransmitted; class 2, parenterally transmitted, i.e., Hepatitis C);Norwalk and related viruses, and astroviruses.

5.3.2 Bacteria

In certain embodiments of the invention, the infectious agent is abacterium. Non-limiting examples of bacteria encompassed by the methodsof the invention include Mycobacteria, Streptococcus, Staphylococcus,Pseudomonas, Salmonella, Neisseria, and Listeria. In a preferredembodiment, the bacteria is Neisseria gonorrhea, M. tuberculosis, M.leprae, Listeria monocytogenes, Streptococcus pneumoniae, S. pyogenes,S. agalactiae, S. viridans, S. faecalis, or S. bovis

Other examples of bacteria contemplated include, but are not limited to,Gram positive bacteria (e.g., Listeria, Bacillus such as Bacillusanthracis, Erysipelothrix species), Gram negative bacteria (e.g.,Bartonella, Brucella, Campylobacter, Enterobacter, Escherichia,Francisella, Hemophilus, Klebsiella, Morganella, Proteus, Providencia,Pseudomonas, Salmonella, Serratia, Shigella, Vibrio, and Yersiniaspecies), spirochete bacteria (e.g., Borrelia species including Borreliaburgdorferi that causes Lyme disease), anaerobic bacteria (e.g.,Actinomyces and Clostridium species), Gram positive and negative coccalbacteria, Enterococcus species, Streptococcus species, Pneumococcusspecies, Staphylococcus species, Neisseria species.

Additional non-limiting examples of specific infectious bacteria includeHelicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria avium, M. intracellulare, M. kansaii, M. gordonae, M.africanum, Staphylococcus aureus, Neisseria meningitidis, Haemophilusinfluenzae, Bacillus antracis, corynebacterium diphtheriae,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturellamultocida, Fusobacterium nucleatum, Streptobacillus moniliformis,Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, andActinomyces israelli.

5.3.3 Parasites

Parasitic diseases that can be treated or prevented by the methods ofthe invention include, but are not limited to, amebiasis, malaria,leishmania, coccidia, giardiasis, cryptosporidiosis, toxoplasmosis,trypanosomiasis, schistosomiasis, and filariasis. In a preferredembodiment, the parasiste is Malaria, Leishmaniases (cutaneous,visceral, and mucocutaneous leishmaniasis). Trypanosoma cruzi, orTheileria parva.

Also encompassed are infections by various worms, such as but notlimited to ascariasis, ancylostomiasis, trichuriasis, strongyloidiasis,toxoccariasis, trichinosis, onchocerciasis filaria, and dirofilariasis.Also encompassed are infections by various flukes, such as but notlimited to schistosomiasis, paragonimiasis, and clonorchiasis.

Further non-limiting examples of parasites include Plasmodium spp.,Toxoplasma gondii, Babesia spp., Trichinella spiralis, Entamoebahistolytica, Giardia lamblia, Enterocytozoon bieneusi, Naegleria,Acanthamoeba, Trypanosoma rhodesiense and Trypanosoma gambiense,Isospora spp., Cryptosporidium spp, Eimeria spp., Neospora spp.,Sarcocystis spp., and Schistosoma spp.

5.4 Pharmaceutical Compositions and Use

The present invention provides methods for treating or preventing aninfectious disease in a subject, the methods comprising administering tothe subject at least two compositions, each comprising at least oneantigenic molecule having one or more epitopes of the infectious agentwhich causes the disease. The antigenic molecules may be polypeptides orproteins, preferably glycosylated polypeptides or proteins. Thepolypeptides or proteins may be purified from an organism or may beproduced, for example using recombinant technology. The antigenicmolecules may also comprise lipoproteins, peptidoglycans,protein-conjugated capsular polysaccharides, capsular polysaccharides,toxoids, an inactivated infectious agent, or portions the inactivatedagent, such as extracts or subunits of the agent (e.g., a virus,bacterium, parasite, or protozoan)

The administering is performed such that each composition isadministered to a different site of the subject. In a preferredembodiment, each site is or substantially drains to, an anatomicallydistinct bodily part selected from a lymph node, a group of lymph nodes,a nonencapsulated cluster of lymphoid tissue, or the spleen. In anotherpreferred embodiment, the distance between each site is greater than thedistance between any two anatomically distinct lymph nodes or groups oflymph nodes nearest each site. The same or a separate route ofadministration may be used for each composition. Preferably, the routeof administration is chosen in order to target a composition to aparticular site.

Non-limiting examples of methods of administration, formulations,effective amounts, dosages, and kits are provided by Sections 5.4.1 to5.4.4. The present methods also encompass administering the compositionsof the invention in combination with one or more therapeutic agents thataid in the prevention or treatment of infectious diseases. Theseembodiments, as well as specific examples of such therapeutic agents areprovided by Section 5.5.

5.4.1 Methods of Administration

Any suitable route of administration is encompassed by the methods ofthe invention, e.g. intradermal, subcutaneous, intravenous,intramuscular, or mucosal. Mucosal routes of administration include, butare not limited to, oral, rectal, vaginal, and nasal administration. Ina preferred embodiment, at least one composition is administeredtransdermally, intradermally, subcutaneously, orally, rectally,vaginally or by inhalation.

Preferably, the route of administration is selected to target acomposition to a particular site, for example, by injection directlyinto a lymph node or a lymph node cluster, by oral administration totarget the lymph nodes of the stomach, by anal administration to targetthe lymph nodes of the rectum, by inhalation or aerosol to target thelymph nodes of the lungs, or by any other suitable route ofadministration.

The methods of the invention provide for the administration of aplurality of compositions. In one embodiment, the number of compositionsadministered is in the range of 2-12, 2-10, 2-8, 2-6, 2-5, or 2-3.Preferably, the number of compositions administered is in the range of2-5. In one embodiment, the number of compositions administered is 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12. In a preferred embodiment, the numberof compositions administered is 2, 3, 4, or 5.

Preferably, each composition is administered at substantially the sametime, for example, within one to eight hours or during the same doctor'svisit. In one embodiment, each composition is administered within one totwo hours, within one to three hours, within one to four hours, orwithin one to five hours.

In another embodiment, the methods of the invention further compriseadministering at least one composition at a separate time, prior to theadministration of the other compositions. For example, at least onecomposition is administered 3 to 14 days before the other compositionsare administered. In a preferred embodiment, at least one composition isadministered 6 days before the other compositions are administered.Preferably, the composition administered at an earlier time comprisesone or more subdominant epitopes and at least one of the compositionsadministered at the later time comprises one or more dominant epitopes,and all of the compositions administered at the later time areadministered together, at substantially the same time. In particularembodiments, the first composition is administered 3 days, 4 days, 5days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 daysor 14 days before the other compositions are administered. In anotherembodiment, the first composition is administered 3-10 days, 4-10 days,5-10 days, or 6-10 days before the other compositions are administered.In certain embodiments, at least one composition is administered 1month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8months, 9 months, 10 months, 11 months, or 12 months before the othercompositions are administered.

The compositions of the invention may also be administered prior to,concurrently with, or subsequent to the administration of one or moreadjuvants. Adjuvants contemplated include, but are not limited to,oil-based adjuvants, CpG oligonucleotides, aluminum salt adjuvants,calcium salt adjuvants, emulsions and surfactant-based formulations. Theadjuvants contemplated for administration prior to, concurrently with,or subsequent to the administration of the composition of the inventionare described in more detail in Section 5.4.2 in connection withformulations. It is envisioned that the adjuvants discussed in Section5.4.2 may optionally be administered separately from the compositions,rather than in formulation with them as described in that section.

5.4.2 Formulations

Pharmaceutical compositions comprising the compositions of theinvention, and their physiologically acceptable salts and solvates, canbe formulated using one or more physiologically acceptable carriers orexcipients. The formulations are preferably for intradermal orsubcutaneous administration, but can be for administration by othermeans such as by inhalation or insufflation (either through the mouth orthe nose), oral, buccal, parenteral, vaginal, or rectal. Preferably, thecompositions are formulated to provide increased chemical stability ofthe antigenic molecules during storage and transportation. For example,in one embodiment, the antigenic molecules comprise polypeptides and theformulation prevents or reduces oligomerization of the polypeptides. Inanother example, the formulation prevents or reduces oxidation of theamino acid residues of the polypeptides. The formulations may belyophilized or liquid formulations.

In one embodiment, the compositions are formulated for injection. In apreferred embodiment, the compositions are sterile lyophilizedformulations, substantially free of contaminating cellular material,chemicals, virus, or toxins. In a particular embodiment, formulationsfor injection are provided in sterile single dosage containers. Theformulations may or may not contain an added preservative. Liquidformulations may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. In one embodiment, theformulation comprises liposomes.

In one embodiment, the compositions further comprise one or moreadjuvants. Adjuvants may comprise any number of delivery systems, forexample, mineral salts, surface active agents, synthetic microparticles,oil-in-water emulsions, immunostimulatory complexes, liposomes,virosomes, and virus-like particles. Adjuvants further comprises one ormore potentiators of the immune response such as microbial derivatives(e.g., bacterial products, toxins such as cholera toxin and heat labiletoxin from E. coli, lipids, lipoproteins, nucleic acids, peptidogylcans,carbohydrates, peptides), cells, cytokines, (e.g., dendritic cells,IL-12, and GM-CSF), hormones, and small molecules. Adjuvantscontemplated include, but are not limited to, oil-based adjuvants (e.g.,Freund's adjuvant), CpG oligonucleotides (see Klinman 2003 Expert Rev.Vaccines 2:305-15) aluminum salt adjuvants, calcium salt adjuvants,emulsions and surfactant-based formulations (e.g., MF59, ASO2,montanide, ISA-51, ISA-720, and QA21). For a review of improvements invaccine adjuvants, see Pashine et al. 2005, Nature Med. 11(4):S63-S68.

5.4.3 Effective Amounts and Dosages

An effective amount of the compositions of the invention is the amountsufficient to reduce the severity of an infectious disease or one ormore symptoms thereof, the amount sufficient to reduce the duration ofthe disease, the amount sufficient to ameliorate one or more symptoms ofthe disease, the amount sufficient to prevent the incidence oradvancement of the disease or the amount sufficient to enhance orimprove the therapeutic effect(s) of another therapy or therapeuticagent.

In one embodiment, the effective amount of the compositions of theinvention is the amount sufficient to produce an antibody secreting Bcell or cytotoxic T cell mediated immune response directed against oneor more molecules of each composition administered to a subject. Theability of the molecules of a composition to elicit an immune responsecan be determined using any routine method available to those of skillin the art. Non-limiting examples of such methods are provided inSection 5.6. In one embodiment, the effective amount of each compositionis the amount sufficient to produce a cytotoxic T cell response in thesubject as measured, for example, by a mixed lymphocyte T cell assay.

In a preferred embodiment, the effective amount of each composition isan amount sufficient to mitigate or avoid the immunodominance of anepitope of an antigenic molecule of any of the compositions.

In one embodiment, the effective amount of each composition administeredat a particular site of the subject is that which delivers an amount ofantigenic molecules in the range of 1 to 1000 micrograms. In a specificembodiment, the amount of antigenic molecules is in the range of 1 to100 micrograms, 1 to 200 micrograms, 1 to 300 micrograms, 1 to 400micrograms, 1 to 500 micrograms, 1 to 600 micrograms, 1 to 700micrograms, 1 to 800 micrograms, or 1 to 900 micrograms. In anotherspecific embodiment, the amount of antigenic molecules is in the rangeof 1 to 10 micrograms, 1 to 20 micrograms, 1 to 30 micrograms, 1 to 40micrograms, 1 to 50 micrograms, 1 to 60 micrograms, 1 to 70 micrograms,1 to 80 micrograms, or 1 to 90 micrograms. Each composition may comprisethe same or a different amount of antigenic molecules. In a preferredembodiment, the amount of antigenic molecules of a compositioncomprising dominant epitopes is less than that of the othercompositions. Preferably, the total amount of antigenic moleculesadministered to a subject does not exceed 5 milligrams, and mostpreferably the total amount does not exceed 2 milligrams, where theadministering of each composition is at substantially the same time.

5.4.4 Kits

The invention provides a pharmaceutical pack or kit for carrying out themethods or therapeutic regimens of the invention. In one embodiment, thekit comprises in separate containers at least two compositions, eachcomposition comprising one or more epitopes of an infectious agent orstrain thereof. In another embodiment, each composition comprises atleast one purified antigenic molecule, and each purified antigenicmolecule comprising one or more epitopes of one or more infectiousagents or strains thereof, the epitopes having an epitopic variance ofbetween 0.05 and 0.50.

In another embodiment, the kit further comprises in one or moreadditional containers an anti-viral agent, an anti-bacterial agent, acytokine, or an adjuvant.

The composition in each container may be in the form of apharmaceutically acceptable solution, e.g., in combination with sterilesaline, dextrose solution, or buffered solution, or otherpharmaceutically acceptable sterile fluid. Alternatively, thecomposition may be lyophilized or desiccated; in this instance, the kitoptionally further comprises in a separate container a pharmaceuticallyacceptable solution (e.g., saline, dextrose solution, etc.), preferablysterile, to reconstitute the composition to form a solution forinjection purposes.

In another embodiment, the kit further comprises one or more reusable ordisposable device(s) for administration (e.g, syringes, needles,dispensing pens), preferably packaged in sterile form, and/or a packagedalcohol pad. Instructions are optionally included for administration ofthe compositions by a clinician or by the patient. The kit may alsocomprise other materials, e.g., metal or plastic foil, such as a blisterpack.

5.5 Combination Therapy

In certain embodiments, the compositions of the invention areadministered in combination with one or more therapeutic agents that aidin the prevention or treatment of infectious diseases. In a particularembodiment, the agent is an antigen presenting cell, a cytokine, anantibiotic, an antiviral compound, an antiprotozoal compound, anantifungal compound, or an antihelminthic compound.

In one embodiment, the infectious disease is caused by the dengue virusand the compositions of the invention are administered in combinationwith one or more antisense nucleic acids which suppress or inhibit theexpression of one or more dengue genes. The nucleic acids may bedeoxyribonucleic acids or ribonucleic acids, or a combination thereof.

In one embodiment, the infectious disease is Lassa Fever and thecompositions of the invention are administered in combination withribavirin.

In one embodiment, the infectious disease is caused by a herpes virusand the compositions of the invention are administered in combinationwith one or more antiviral agents selected from the group consisting ofacyclovir, brivudin, cidofovir, famciclovir, foscarnet, ganciclovir,idoxuridine, trifluridine, valaciclovir, and vidarabine.

In one embodiment, the infectious disease is caused by a hepatitis B orC virus and the compositions of the invention are administered incombination with one or more antiviral agents selected from the groupconsisting of interferon, adefovir dipivoxil, and lamivudine.

In one embodiment, the infectious disease is caused by a hepatitis Bvirus and the compositions of the invention are administered incombination with one or more antiviral agents selected from the groupconsisting of acyclovir, famciclovir, and ganciclovir.

In one embodiment, the infectious disease is caused by a rhinovirus andthe compositions of the invention are administered in combination withone or more antiviral agents selected from the group consisting ofpirodavir and pleconaril.

In one embodiment, the compositions of the invention are administeredalong with antigen presenting cells (“APCs”) which have been sensitizedaccording to art-recognized methods with one or more antigenic moleculesof a composition. In accordance with this embodiment, the antigenicmolecule-pulsed APCs serve as adjuvants for vaccination as described,for example, by Martin-Fontecha et al. 2003 J. Exp. Med. 198, 615-621.APCs, including but not limited to macrophages, dendritic cells andB-cells, can be obtained by any of various methods known in the art.Preferably, the APCs are obtained by production in vitro from stem andprogenitor cells from human peripheral blood or bone marrow and mostpreferably the APCs are obtained from the subject or are autologous tothe subject.

In one embodiment, the compositions of the invention comprise APCssensitized with one or more antigenic molecules of an infectious agent.In another embodiment, the compositions of the invention areadministered at substantially the same time as APCs sensitized with oneor more antigenic molecules of a composition. In another embodiment, theAPCs are administered prior to the compositions of the invention. TheAPCs are preferably administered at the same site as the compositioncomprising the antigenic molecules with which the APCs were sensitized.

5.5.1 Cytokines

The compositions of the invention may optionally be administered incombination with one or more cytokines. In one embodiment, thecompositions of the invention comprise one or more cytokines. In apreferred embodiment, at least one cytokine is an interleukin or aninterferon. In a particular embodiment, at least one cytokine is aninterleukin selected from the group consisting of IL-1α, IL-1β, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15,and IL-18. In another particular embodiment, at least one cytokine is aninterferon selected from IFNα, IFNβ, and IFNγ.

5.5.2 Antiviral Agents

Antiviral agents that can be used in combination with the compositionsof the invention include, but are not limited to, non-nucleoside reversetranscriptase inhibitors, nucleoside reverse transcriptase inhibitors,protease inhibitors, and fusion inhibitors. In one embodiment, theantiviral agent is selected from the group consisting of amantadine,oseltamivir phosphate, rimantadine, and zanamivir. In one embodiment,the antiviral agent is a non-nucleoside reverse transcriptase inhibitorselected from the group consisting of delavirdine, efavirenz, andnevirapine. In one embodiment, the antiviral agent is a nucleosidereverse transcriptase inhibitor selected from the group consisting ofabacavir, didanosine, emtricitabine, emtricitabine, lamivudine,stavudine, tenofovir DF, zalcitabine, and zidovudine. In one embodiment,the antiviral agent is a protease inhibitor selected from the groupconsisting of amprenavir, atazanavir, fosamprenav, indinavir, lopinavir,nelfinavir, ritonavir, and saquinavir. In one embodiment, the antiviralagent is a fusion inhibitor such as enfuvirtide.

Additional, non-limiting examples of antiviral agents for use incombination with the methods of the invention include the following:rifampicin, nucleoside reverse transcriptase inhibitors (e.g., AZT, ddI,ddC, 3TC, d4T), non-nucleoside reverse transcriptase inhibitors (e.g.,delavirdine efavirenz, nevirapine), protease inhibitors (e.g.,aprenavir, indinavir, ritonavir, and saquinavir), idoxuridine,cidofovir, acyclovir, ganciclovir, zanamivir, amantadine, andpalivizumab. Other examples of anti-viral agents include but are notlimited to acemannan; acyclovir; acyclovir sodium; adefovir; alovudine;alvircept sudotox; amantadine hydrochloride (SYMMETREL™); aranotin;arildone; atevirdine mesylate; pyridine; cidofovir; cipamfylline;cytarabine hydrochloride; delavirdine mesylate; desciclovir; didanosine;disoxaril; edoxudine; enviradene; enviroxime; famciclovir; famotinehydrochloride; fiacitabine; fialuridine; fosarilate; foscamet sodium;fosfonet sodium; ganciclovir; ganciclovir sodium; idoxuridine; kethoxal;lamivudine; lobucavir; memotine hydrochloride; methisazone; nevirapine;oseltamivir phosphate (TAMIFLU™); penciclovir; pirodavir; ribavirin;rimantadine hydrochloride (FLUMADINE™); saquinavir mesylate; somantadinehydrochloride; sorivudine; statolon; stavudine; tilorone hydrochloride;trifluridine; valacyclovir hydrochloride; vidarabine; vidarabinephosphate; vidarabine sodium phosphate; viroxime; zalcitabine; zanamivir(RELENZA™); zidovudine; and zinviroxime.

5.5.3 Antibacterial Agents

Antibacterial agents, including antibiotics, that can be used incombination with the compositions of the invention include, but are notlimited, to aminoglycoside antibiotics, glycopeptides, amphenicolantibiotics, ansamycin antibiotics, cephalosporins, cephamycinsoxazolidinones, penicillins, quinolones, streptogamins, tetracyclins,and analogs thereof.

In one embodiment, the antibacterial agent is selected from the groupconsisting of ampicillin, amoxicillin, ciprofloxacin, gentamycin,kanamycin, neomycin, penicillin G, streptomycin, sulfanilamide, andvancomycin.

In one embodiment, the antibacterial agent is selected from the groupconsisting of azithromycin, cefonicid, cefotetan, cephalothin,cephamycin, chlortetracycline, clarithromycin, clindamycin, cycloserine,dalfopristin, doxycycline, erythromycin, linezolid, mupirocin,oxytetracycline, quinupristin, rifampin, spectinomycin, and trimethoprim

Additional, non-limiting examples of antibacterial agents for use incombination with the methods of the invention include the following:aminoglycoside antibiotics (e.g., apramycin, arbekacin, bambermycins,butirosin, dibekacin, neomycin, neomycin, undecylenate, netilmicin,paromomycin, ribostamycin, sisomicin, and spectinomycin), amphenicolantibiotics (e.g., azidamfenicol, chloramphenicol, florfenicol, andthiamphenicol), ansamycin antibiotics (e.g., rifamide and rifampin),carbacephems (e.g., loracarbef), carbapenems (e.g., biapenem andimipenem), cephalosporins (e.g., cefaclor, cefadroxil, cefamandole,cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide, andcefpirome), cephamycins (e.g., cefbuperazone, cefinetazole, andcefminox), folic acid analogs (e.g., trimethoprim), glycopeptides (e.g.,vancomycin), lincosamides (e.g., clindamycin, and lincomycin),macrolides (e.g., azithromycin, carbomycin, clarithomycin,dirithromycin, erythromycin, and erythromycin acistrate), monobactams(e.g., aztreonam, carumonam, and tigemonam), nitrofurans (e.g.,furaltadone, and furazolium chloride), oxacephems (e.g., flomoxef, andmoxalactam), oxazolidinones (e.g., linezolid), penicillins (e.g.,amdinocillin, amdinocillin pivoxil, amoxicillin, bacampicillin,benzylpenicillinic acid, benzylpenicillin sodium, epicillin,fenbenicillin, floxacillin, penamccillin, penethamate hydriodide,penicillin o benethamine, penicillin 0, penicillin V, penicillin Vbenzathine, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium), quinolones and analogs thereof (e.g.,cinoxacin, ciprofloxacin, clinafloxacin, flumequine, grepagloxacin,levofloxacin, and moxifloxacin), streptogramins (e.g., quinupristin anddalfopristin), sulfonamides (e.g., acetyl sulfamethoxypyrazine,benzylsulfamide, noprylsulfamide, phthalylsulfacetamide,sulfachrysoidine, and sulfacytine), sulfones (e.g., diathymosulfone,glucosulfone sodium, and solasulfone), and tetracyclines (e.g.,apicycline, chlortetracycline, clomocycline, and demeclocycline).Additional examples include cycloserine, mupirocin, tuberin amphomycin,bacitracin, capreomycin, colistin, enduracidin, enviomycin, and 2,4diaminopyrimidines (e.g., brodimoprim).

5.5.4 Antiprotozoal and Antiparasitic Agents

Many examples of agents that can be used in combination with thecompositions of the invention to treat protozoal or parasitic diseasesare known in the art and include, but are not limited to, quinines,chloroquine, mefloquine, proguanil, pyrimethamine, metronidazole,diloxanide furoate, tinidazole, amphotericin, sodium stibogluconate,trimoxazole, and pentamidine isetionate. Specific non-limiting examplesinclude mebendazole, levamisole, niclosamide, praziquantel, albendazole,ivermectin, diethylcarbamazine, thiabendazole, acedapsone, amodiaquinehydrochloride, amquinate, arteflene, chloroquine, chloroquinehydrochloride, chloroquine phosphate, cycloguanil pamoate, enpirolinephosphate, halofantririe hydrochloride, hydroxychloroquine sulfate,mefloquine hydrochloride, menoctone, mirincamycin hydrochloride,primaquine phosphate, pyrimethamine, quinine sulfate, and tebuquine.

5.6 Methods of Measuring and Monitoring Immunodominance

The methods of the present invention are particularly useful formitigating the effects of immunodominance in the context of therapeuticor prophylactic vaccination against one or more infectious diseases. Theefficacy of the therapeutic or prophylactic methods described herein mayvary depending on such factors as the epitopes of the moleculescomprising the compositions to be administered, the sites chosen foradministration, the time of administration, and the particularinfectious agent to be targeted. Accordingly, the present invention alsoprovides methods for determining whether a vaccine compositionsuppresses immunodominance in a subject so that the methods may beoptimized for a particular subject, population of subjects, or for oneor more infectious diseases. These methods can also be used to monitorthe efficacy of therapeutic or prophylactic vaccination in a subject orin a population of subjects.

The methods for measuring and monitoring immunodominance in a subjectcomprise administering to the subject a plurality of compositions, eachcomposition being administered to a different site of the subjectaccording to the methods of the invention and each compositioncomprising at least one antigenic molecule as described herein, andmeasuring an immune response to the antigenic molecule or molecules ofeach composition so administered. Immunodominance has been suppressed inthe subject if the ratio between the immune response of the mostimmunogenic composition and the least immunogenic composition is reducedcompared to the ratio obtained with administration of all of thecompositions at a single site. In one embodiment, the ratio is reducedby 10%-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90% or90-100%. In another embodiment, the ratio is reduced by about 20%, about40%, about 60%, about 80%, about 85%, about 90%, or about 95%. In apreferred embodiment, the ratio is reduced by about 50-99%.

Any routine assay for measuring an immune response to an antigenicmolecule may be used in accordance with this embodiment. Preferably, theassay determines a cytotoxic T cell response to the antigenic moleculesof each composition. In a preferred embodiment, the cytotoxic T cellresponse is determined by a tetramer assay or by measuring the cytokineprofiles of responding cells, for example using and ELISPOT assay or anintracellular cytokine staining assay. In another embodiment, theresponse is measured by assaying the ability of T cells obtained fromthe subject to specifically lyse a population of target cells expressingor coated with the antigenic molecules of each composition administeredto the subject. Non-limiting examples of methods for measuring theimmune response elicited against the antigenic molecules of eachcomposition are provided below for purposes of illustration only, andare not intended to limit the kinds of assays for measuring an immuneresponse which may be used in the methods of the invention. For asummary assays useful for T cell responses see Robinson and Amara, 2005Nature Med. 11(4):S25-32, and references infra.

5.6.1 Tetramer Staining Assay

The ability of the compositions administered according to the methods ofthe invention to elicit a cellular immune response, e.g., a CD4+ or aCD8+ T cell response, is preferably measured with a tetramer stainingassay. This assay identifies antigen-specific T cells (see Altman etal., 1996, Science 274: 94-96; Savage et al., 2002 Br. J. Cancer86:1336-42; Choi et al., 2003 J. Immunol. 171:5116-23). For example,tetramers are produced by multimerizing a MHC molecules complexed withthe antigenic molecules, and labeled, for example, by complexing tostreptavidin or a fluorescent molecule. The labeled tetramer solution isthen mixed with a population of lymphocytes or peripheral bloodmononuclear cells obtained from a subject treated according to themethods of the invention. T cells can be identified, for example, bylabeling with anti-CD8+ fluorescent antibodies. The population of cellsthat are both CD8+ and which bind to the labeled tetramers can bedetected, for example, by dual color flow cytometry.

5.6.2 The MLTC or Chromium Release Assay

An immune response elicited according to the methods of the inventioncan also be measured with a conventional mixed lymphocyte T cell assay,also referred to as a ⁵¹Cr release assay, using cells which have beenpulsed with the antigenic molecule or molecules of the compositions aslysis targets. Briefly, the compositions of the invention areadministered to a mammalian subject such as a mouse according to themethods described herein. Appropriate negative controls include, forexample, administration of the same compositions at a single site,instead of at multiple sites, or administration of the compositionswithout antigenic molecule or molecules (e.g., a sham vaccination).Following a period of time after the administration of the compositions,the spleens of the vaccinated animals are removed and the lymphocytesare cultured in vitro according to routine methods. To measure theprimary response, the period of time is generally from 3 to 12 days,preferably 4, 8, or 10 days for a mouse. To measure the secondary, e.g,memory, response, a longer time period is used, for example 30 days, 45days, or 60 days. The cultured lymphocytes may optionally bere-stimulated in vitro by the addition of the antigenic molecule ormolecules of the compositions that were administered. It may bedesirable to include an amount of secondary mixed lymphocyte culturesupernatant in the culture medium as a source of T cell growth factors(See, Glasebrook, et al., 1980, J. Exp. Med. 151:876). For example, theculture medium may comprise 30-40% of such supernatant.

After culturing in vitro for a period of time, usually from 3 to 7 days,the lymphocytes are tested for their ability to specifically lyse targetcells coated with or expressing the antigenic molecule or molecules ofthe compositions that were administered. Specific lysis is measured, forexample, with a ⁵¹Cr-release assay (see Palladino, et al., 1987, CancerRes. 47:5074-5079 and Blachere et al., 1993, J. Immunotherapy14:352-356). In this assay, the mixed lymphocyte culture is added tolabeled target cells. The target cells are pre-labeled with theradioactive isotope by incubating the cells in culture medium containingthe isotope. Typically, a number of different ratios of effector cellsto target cells is used, e.g., in the range of 1:1 to 40:1. Lysis ismeasured as a function of ⁵¹Cr-release. Each assay point comprises aneffector cell to target cell ratio, typically performed in triplicate.Appropriate positive and negative controls for ⁵¹Cr-release are, forexample, target cells alone to measure spontaneous ⁵¹Cr-release, andcells lysed with detergent to measure 100% release. The amount of ⁵¹Crreleased into the supernatant is measured by routine methods, forexample by a gamma counter. Specific lysis is measured as theradioactivity, e.g., in counts per minute, or “cpm,” in the test sampleminus radioactivity in the negative control (spontaneously released)divided by the radioactivity in the positive control (total detergentreleased) less the negative control. Optionally, in order to block theMHC class I cascade a concentrated hybridoma supernatant derived fromK-44 hybridoma cells (an anti-MHC class I hybridoma) is added to thetest samples to a final concentration of 12.5%.

5.6.3 CD4+ or CD8+ T Cell Proliferation Assay

The ability of the compositions administered according to the methods ofthe invention to elicit a cellular immune response can also be measuredas the ability to promote CD4+ or CD8+ T cell proliferation in vitrofollowing exposure of peripheral blood mononuclear cells to theantigenic molecule or molecules of the compositions with which a subjecthas been vaccinated according to the methods of the invention. Primarycells are obtained from spleen, fresh blood, or CSF and purified usingroutine methods; for example, as described by Kruse and Sebald, 1992,EMBO J. 11: 3237 3244. The cells are incubated for a period of time,e.g., 5-12 days, with the antigenic molecule or molecules, optionallywith added adjuvant or antigen presenting cells which may be added tothe culture 24 to 48 hours prior to the assay. The proliferation of thecells is then measured using any of the numerous art-recognized methodsfor measuring cell proliferation. For example, proliferation can bemeasured by radiometric assays such as tritiated thymidineincorporation, by colorimetric assays such as the MTT assay, or byfluorescence assays such as those utilizing fluorescently labelednucleotides. In another example, proliferation is measured using thecarboxyl fluorescein diacetate succinimidyl (CFSE) assay which utilizesflow cytometry for detection of proliferation and cell type.

5.6.4 Antibody Response Assay

The ability of the compositions administered according to the methods ofthe invention to elicit a cellular immune response can also bedetermined by measuring antibodies produced in response to vaccinationwith the compositions. For example, plates are coated with the antigenicmolecule or molecules which comprise the compositions used in thevaccine and incubated with plasma or CSF from a vaccinated subject (suchas a model mouse or a human patient). The presence of antibodies whichhave bound to the antigenic molecule or molecules is detected usingrouting methods, for example by incubation with a secondary antibodysuch as a sheep anti-mouse or anti-human immunoglobulin, as appropriate,conjugated with a detectable label or an enzyme such as horseradishperoxidase. The amount of secondary antibody which specifically binds tothe plates is determined using a method appropriate for the detection ofthe label or the presence of the conjugated enzyme.

5.6.5 Cytokine Detection Assay

The ability of the compositions administered according to the methods ofthe invention to elicit a cellular immune response can also bedetermined indirectly, through the detection of cytokines known to beproduced by activated T cells, for example, using an intracellularcytokine staining assay. Thus, the CD4+ and CD8+ T cell response can bemeasured by detection and quantitation of the levels of specificcytokines. For example, intracellular cytokines can be measured using anIFN-gamma detection assay. In this method, peripheral blood mononuclearcells from a subject treated according to the methods described hereinare stimulated with the antigenic molecule or molecules of thecompositions administered to the subject. T cells are identified byincubating the cells with T cell-specific labeled antibodies. Similarly,IFN-gamma is detected by incubating the cells with labeled antibodiesreactive with IFN-gamma. The labeled cells are detected and quantitated,for example, by flow cytometry.

Alternatively, a filter immunoassay, such as the enzyme-linkedimmunospot assay (ELISPOT) assay, can be used to detect specificcytokines surrounding a T cell. For example, a nitrocellulose-backedmicrotiter plate is coated with a purified cytokine-specific primaryantibody, i.e., anti-IFN-gamma, and the plate is blocked to avoidbackground due to nonspecific binding of other proteins. A sample ofmononuclear blood cells obtained from a subject treated according to themethods of the invention is diluted onto the wells of the microtitreplate. A labeled secondary anti-cytokine antibody is added and detectedusing means appropriate for the particular label. Cytokine-secretingcells appear as “spots” which can be quantitated by visual, microscopic,or electronic detection methods.

6. EXAMPLE Sculpting the Immunological Response to Dengue Fever byPolytopic Vaccination

The present invention is not to be limited in scope by the specificembodiments described herein. Various modifications of the invention inaddition to those described herein will become apparent to those skilledin the art from the foregoing description. The following exampleillustrates the theoretical basis of the invention using the denguevirus. However, the methods of the present invention are believed to begeneralizable to any infectious agent against which an immune responseis prophylactic or therapeutic.

The twin challenges of immunodominance and heterologous immunity havehampered discovery of an effective vaccine against all four strains ofdengue virus. This example provides a model that describes how T cellcompetition and selection impede effective vaccine design. The model'saccuracy is evidenced by its ability to predict dengue vaccine clinicaltrial data. The model further indicates two novel methods forvaccination against dengue: polytopic injection and subdominant epitopepriming.

Dengue virus infections are a serious cause of morbidity and mortalityin most tropical and subtropical areas of the world (Gubler, 1998;Kurane and Takasaki, 2001; Gibbons and Vaughn, 2002). There are fourstrains of dengue, and immunity built up after infection by one strainof dengue virus protects only modestly or even negatively againstreinfection by the other strains (Mongkolsapaya et al. 2003; Rothman,2004; Klenerman and Zinkernagel, 1998). Thus, an effective vaccine fordengue must induce protective immunity against all four strains. Todate, no such vaccine has been developed. The immunological epitopes ofthe four strains of the dengue virus are closely related but differ insequence, and it is believed that the immunological response to eachstrain is largely to a single epitope (Mathew et al. 1996; Zivny et al.1995). These epitope sequence differences affect the quality of theeffector response. For example, simultaneous exposure to all fourepitopes reduces the quality of the immune response to some of thestrains, by a mechanism that is not understood. (Rothman et al. 2001;Gubler 1998). Most studies suggest that CD8+ T cell receptors (TCRs)specific for dominant epitopes suppress response to other epitopes, dueto resource competition, homeostasis, apoptosis, and reduction of viralload (Yewdell and Bennink, 1999; Freitas and Rocha, 2000; Nara andGarrity, 1998).

In this example, a generalized NK, or spin glass, model of T celloriginal antigenic sin and immunodominance is used (Deem and Lee, 2003;Park and Deem, 2004). The model captures the stochastic nature of humanimmunity and realistic recognition characteristics between TCRs andvirus at the sequence level. For example, the model addresses thecompetition of cross-active T cells to the four strains of dengue thatleads to the selection of effector and memory T cells. The model is thusa simplified virtual immune system focusing on the competitive selectiondynamics of CD8+ TCRs. As such, it is useful as a tool for T cellvaccine design. The results obtained with the model suggest thatpolytopic vaccination, that is, vaccination with a plurality ofcompositions, each composition being administered to a different site ofthe subject, wherein each composition comprises at least one antigenicmolecule, wherein at least one antigenic molecule in each compositioncomprises one or more epitopes of the same infectious agent or a strainthereof, and wherein the one or more molecules of each compositioncomprise in aggregate a set of epitopes distinct from that of any othercomposition so administered, is efficacious for the treatment andprevention of infectious diseases, particularly diseases caused bymulti-strain infectious agents.

6.1 Specific Lysis

The model captures the stochastic nature of human immunity and realisticrecognition characteristics between TCRs and virus at the sequencelevel. Agreement within experimental error bars is observed betweenpredictions from the model and observations from clinical vaccine trials(see FIG. 1). We emphasize that this result is not a fit, as the modelwas calibrated to the altered peptide lingand data (Park and Deem, 2004)before this prediction was made.

6.2 Original Antigenic Sin and Immunodominance

We first discuss how cross-immunity to the four strains of dengue shapesthe T cell repertoire differently under variant immunological histories.FIG. 2 a shows the response to a second antigen if the exposure to afirst antigen exists (solid line) or not (dashed line) as a function ofthe difference between the first and second antigen, p_(epitope). Whenthe difference is small, the exposure to a first antigen leads to ahigher clearance probability, Z, than without exposure. For a largedifference, the antigen encountered in the first exposure isuncorrelated with that in the second exposure, and so immune systemmemory does not play a role. Interestingly, the immunological memoryfrom the first exposure actually gives worse protection, a lower z, forintermediate differences than would no memory, which is originalantigenic sin (Klenerman and Zinkernagel 1998). The epitopic variationfor dengue lies in this range. During primary infection, T cellpopulations with higher affinity for the infecting strain arepreferentially expanded and enter the memory pool. When exposure toanother strain follows this first exposure, not only is the memory Tcell population at a 100 to 1000 times greater concentration than thenaive T cell population (Janeway et al. 2001), but also the averagebinding constant of the memory T cell population may initially be higherthan that of the naive population, as shown by the round=0 data in FIG.2 b. For dengue, and other diseases, these memory T cells will,therefore, be selectively expanded, even though selection and expansionof the naive population would have produced superior binding constantsof the effector T cells, as shown by the round=10 data in FIG. 2 b. Thatis, memory T cells are expected to perform better against cross-activestrains than would naive TCRs. But our theory shows that for dengue,from FIG. 2 b, this evolved behavior of the immune system is faulted,and naive TCRs can select for superior binding affinity.

We next address how the immune response to one or two dominant strainsof dengue suppresses the response to the other, subdominant strains.This immunodominance can be seen in FIG. 1. The immune response istypically strong to only a few epitopes. The immune response to thesedominant epitopes suppresses the response to the other, subdominantepitopes. This immunodominance is well captured by the generalized NKtheory. Even in the absence of competition of the TCRs for resources,there is immunodominance in the immune response. (FIG. 2 c, leftcolumn). This result shows that immunodominance stems from theheterologous nature of the immune system. It appears inevitable.Vaccination with only a single strain increases the immunodominance(FIG. 2 c, middle columns), as a result of original antigenic sin.

6.3 Sequential Dependence of Response

Priming with the subdominant epitope (FIG. 2 c, right columns, i=1)sculpts a broader immune response and leads to reduced immunodominancein a secondary response than does priming with the dominant epitope(FIG. 2 c, right columns, i=4). The improvement in the response againstthe least-recognized strain is 51%. Epidemiological studies havesuggested that the order of exposure to dengue virus infections isimportant (Sangkawibha et al. 1984). CTLs induced by one strain mayrecognize another strain to a greater extent than the reverse, whichincreases the odds of dengue hemorrhagic fever (DHF) (Spaulding et al.1999; Kurane and Takasaki 2001). We term this the ordering effect. FIG.2 c shows the effect. Our theory suggests that there is nonreciprocalCTL cross-activity among the four dengue virus strains. Initial exposureto the dominant strain will suppress the available TCRs for a subsequentexposure to a subdominant strain more quickly and harmfully than thereverse. Conversely, initial exposure to the subdominant epitope doesnot cause too much suppression against the dominant epitope because ofthe excess of high-affinity TCRs available for the dominant epitope. Ourtheory (FIG. 2 c, right columns, i=1) predicts and epidemiologicalstudies (Sangkawibha et al. 1984) show that primary exposure to thesubdominant DEN-2 followed by secondary exposure to the other strains isthe least likely infection sequence to produce severe dengue disease.Additionally our theory (FIG. 2 c, right columns, i=4) predicts andepidemiological studies (Ferguson et al. 1999; Sangkawibha et al. 1984)also show that primary exposure to other strains of dengue followed bysecondary exposure to the subdominant DEN-2 is the most likely infectionsequence to produce severe disease (Ferguson et al. 1999; Sangkawibha etal. 1984). Our results direct that to construct and administer anoptimal vaccine, the patient history and vaccine ordering effect must beconsidered.

6.4 Polytopic Injection

By physically separating the TCR selection process and reducing thepressure on TCR resource competition within each lymph node, the TCRrepertoire can be sculpted toward the subdominant epitopes, and so thereis a reduction in immunodominance. We term this the polytopic effect.FIGS. 3 and 4 c show this effect. Immunodominance and competitionthrough space imply that to maximize protection and minimize pathologicheterologous immunity and to achieve a long-lasting immune response, anoptimal dengue vaccine should induce a high concentration ofhigh-affinity TCRs against all four strains (Yewdell and Bennink 1999;Rothman et al. 2001; Gubler 2004; Dharakul et al. 1994). In other words,the immune system must be induced to expand TCRs against all dominantand subdominant strains. Larger values of the parameter mixing roundallow for a longer period of independent TCR selection during a typical10-round immune response period and lead to less immunodominance (FIG.3). The parameter mixing round is relative to the T cell division time,which is typically 12-24 hours (Janeway et al. 2001). It is seen thatthe results are not greatly sensitive to the exact value of this lumpedparameter. Some T cells may leave the lymph node before the mixinground, and some may leave after the mixing round, but the average timeto leave is mixing round, and the average behavior is shown in FIG. 3.For the human immune system, the time for T cells to leave the lymphnodes plus the circulation time of the lymph system is in the range 6-10days (Janeway et al. 2001; Fournier, 1999). Within this entirephysiological range, the polytopic effect is beneficial.

6.5 Sculpting the Immunological Response to a Dengue Vaccine

By combining multi-site injection with subdominant epitope priming, apowerful vaccination protocol for sculpting the immune response todengue is achieved (FIG. 4 d). This new protocol is superior tomulti-site injection or subdominant epitope priming alone (FIGS. 4 c and4 b, respectively), both of which are superior to a traditionalfour-component dengue vaccine (FIG. 4 a). Immunological memory andcompetition through time imply that response to a subdominant strain canbe strengthened with prior exposure. Indeed, not only does patienthistory affect the response to vaccination, FIG. 2 a, but also a historycan be imposed by a vaccination sequence.

6.6 Discussion

Original antigenic sin and immunodominance are two sides of one coin.They both stem from the competitive selection of cross-active TCRs.Original antigenic sin acts sequentially through time and arises fromcompetition between memory and naive T cells. Because of originalantigenic sin, prior exposure history is an important factor to theimmune response. Immunodominance acts simultaneously through space on Tcells competing for expansion against multiple, related strains. Becauseof immunodominance, relatedness of strains in a multi-component vaccineis an important factor to the immune response. From FIG. 1 a, we seethat the competitive selection leading to original antigen sin occurs inthe range 0.02<p_(epitope)<0.4, in which dengue lies.

When a single strain disease begins to mutate, the two sides of theheterologous immunity coin become connected. Original antigenic sinimparts to some of the mutants a selective advantage at escaping immunesystem control. Concomitantly, the dominant escape mutants skew the Tcell repertoire toward themselves and away from the subdominant escapemutants (Sette and Fikes 2003). These subdominant mutants can thenproliferate. HIV and cancer are examples of such escape from the immunesystem (McMichael and Rowland-Jones 2001; Khong and Restifo 2002). Inboth cases, immunodominance is a significant barrier to developingeffective vaccines against these multi-strain diseases.

The strategies of polytopic vaccination and subdominant epitope primingapply to vaccination against disease strains expressing multipleepitopes. In this case, the optimal strategy would be to identify andvaccination against all of the significant epitopes in each strain.While epitope based vaccines are becoming the favored approach,polytopic vaccination and subdominant epitope priming will still reduceimmunodominance in whole-strain vaccines if each strain is administeredseparately.

The polytopic injection and subdominant epitope priming ideas applygenerically to multi-strain viral diseases. Competition due to limitedpartial cross-activity is the reason for original antigen sin fordiseases with epitopic variation in the range 0.02<p_(epitope)<0.40.Deleterious competition is why the separate selection that occurs inmulti-site injection leads to less immunodominance and why subdominantepitope priming is useful to achieve a significant number of TCRsresponding to the subdominant epitopes. Our theory captures realisticrecognition characteristics between the TCRs and the virus, the primaryand secondary dynamics due to TCR resource competition, and thestochastic nature of heterologous human immunity. Dengue was unique inour theory because the difference between the epitopes of the fourstrains is roughly p_(epitope)=0.5/9. For strains with largerdifferences but which are still cross-active, as might occur withmulti-strain diseases other than dengue, both subdominant epitopepriming and multi-site injection continue to work effectively (FIG. 5a,d). For example, vaccines against HIV (McMichael and Rowland-Jones2001) or multi-strain cancers (Khong and Restifo 2002) should benefitfrom the proposed approach. When p_(epitope)=1.0/9, such as a variantdengue strain or other multi-strain disease, our strategies can improverecognition of the least-recognized strain by 290% improvement (FIG. 5a,d) The results from our theory not only quantitatively reproduce andexplain immunodominance, but also predict and explain original antigenicsin. From our theory of the ordering and multi-site effects, subdominantepitope priming followed by secondary multi-site injection of epitopesappears to be a promising vaccination strategy for dengue fever andother multi-strain diseases.

6.7 Methods

6.7.1.1 Generalized NK Model

The generalized NK model for the T-cell response considers interactionsbetween the TCR, MHCI, and peptide epitope (Park and Deem, 2004; Deemand Lee, 2003). Parameters in the theory are shown in Table 4. The modelreturns the free energy of binding (U) as a function of the TCR aminoacid sequence (a_(j)) and epitope amino acid sequence (a_(j) ^(pep)).

TABLE 4 Parameter values for the Generalized NK model Para- meter ValueDefinition TCR a_(i) Identity of amino acid at sequence position j M 6Number of secondary structure subdomains N 9 Number of amino acids ineach subdomain L 5 Number of subdomain types (e.g., helices, strands,loops, turns and others) α_(i) 1 ≦ Type of secondary structure for theα_(i) ≦ L i^(th) subdomain K 4 Local interaction range within asubdomain σ_(ai) Local interaction coupling within a subdomain, forsubdomain type α_(i) D 2 Number of interactions between subdomainsσ_(ij) ^(k) Nonlocal interaction coupling between secondary structuresEpitope a_(j) ^(pep) Identity of amino acid of epitope at sequenceposition j N 9 Number of amino acids in epitope TCR- N_(b) 3 Number ofhot-spot amino acids in Epitope the epitope N_(con) 3 Number of aminoacids in TCR that each hot spot interacts with σ_(ij) Interactioncoupling between TCR and epitope σ_(i) ^(k) Interaction coupling betweenTCR secondary structure and epitope Random ω Gaussian random number withzero couplings average and unit standard deviation σ ω_(j) + Value ofcoupling for amino acid i ω_(i)/2 of non-conservative type j

The binding constant is related to the energy byK=e^(a−bU)

We determine the values of a, b in each instance of the ensemble byfixing the geometric average TCR:p-MHCI affinity to be K=10⁴ l/mol andminimum affinity to be K=10² l/mol for the N_(size)=10⁸/10⁵=1000distinct TCRs that respond to one epitope (Hengartner and Zinkernagel2001; Goldrath and Bevan, 1999). This means that for the highestaffinity TCR, K fluctuates between 10⁵ l/mol and 10⁷ l/mol for thedifferent epitopes (van der Merwe and Davis 2003) (see FIG. 6).

Specific lysis is a measure of the probability that an activated T cellwill recognize an antigen presenting cell that is expressing aparticular peptide-MHC complex. It is given by (Park and Deem 2004)

$L = \frac{{zE}/T}{1 + {{zE}/T}}$

where E/T is the effector to target ratio. The quantity z is, therefore,the average clearance probability of one TCR:

$z = {{1/N_{size}}{\sum\limits_{i = 1}^{N_{size}}{{\min\left( {1,{K^{i}/10^{6}}} \right)}.}}}$

6.7.1.2 TCR Selection Dynamics

The naive TCR repertoire is generated randomly from gene fragments. Thisis accomplished by constructing the TCRs from subdomain pools. Fragmentsfor each of the L subdomain types are chosen randomly from 13 of the 100lowest energy subdomain sequences. This diversity mimics the known TCRdiversity, (13×L)^(M)≅10¹¹ (Kesmir, Borghans, and de Boer, 2000). Only 1in 10⁵ naive TCRs responds to any particular antigen, and there are only10⁸ distinct TCRs present at any one point in time in the human immunesystem (Zinkernagel and Hengartner 2001; Goldrath and Bevan 1999), sothe primary response starts with a repertoire of N_(size)=10³ distinctTCRs. A flow diagram of the TCR selection dynamics is shown in FIG. 7.

The T-cell-mediated response is driven by cycles of concentrationexpansion and selection for better binding constants. The primaryresponse increases the concentration of selected TCRs by 1000 fold over10 rounds, with a rough T cell doubling time of 12-24 hours. Thediversity of the memory sequences is 0.5% of that of the naiverepertoire (Arstila et al. 1999). Specifically, 10 rounds of selectionare performed during the primary response, with the top x=58% of thesequences chosen at each round. The T cell division may occur morerapidly than once per day, and the mixing round parameter is measured inthe time scale of T cell divisions. This procedure mimics theconcentration expansion factor of 10³≅2¹⁰ in the primary response andleads to 0.5% diversity of the memory repertoire, because 0.58¹⁰≅0.5%and 10 rounds of doubling leads to a concentration expansion of 2¹⁰=10³.

6.7.1.3 Parameters for Dengue

For each epitope, the sequence, model, and VDJ selection pools differ byp_(epitope) (Deem and Lee, 2003), wherep _(epitope)=(non-conservative+0.5*conservative)amino acid differencesin epitope divided by the total number of amino acids in epitope.

To generate results, an average over many instances of these randomepitope sequences, models, and VDJ selection pools that differ byp_(epitope) is taken. In other words, the different strains of denguewere chosen so that they differ by the requisite p_(epitope). To computeaverage results, four new random epitopes were generated each time. Theinitial TCR repertoire is redetermined for each realization of themodel—this must be done because the U^(sd) that defines the TCRrepertoire is different in each instance of the ensemble.

For dengue, the nonstructural (NS3) protein is an attractive candidatefor a subunit vaccine (Mathew et al. 1996). We consider the observedsingle conservative amino acid change in the epitope (Zivny et al.1995), and so p_(epitope)=0.5/9. Qualitatively similar results areobtained for p_(epitope)=1/9, as might occur for drift strains ofdengue. We assume that for each strain of dengue, only a single epitopeis recognized by the immune system.

6.7.1.4 Polytopic Vaccination

For a polytopic vaccination, the different strains are injected indifferent physical locations and evoke an immune response that evolvesindependently in different lymph nodes until mixing round, after whichthe lymph system is well-mixed. This is modeled by performing a responseagainst each of the four strains independently, with selection among 10³TCRs for each strain, until mixing round. At mixing round, 10³ TCRs arerandomly chosen from the 4×10³ partially evolved TCRs. These 10³ TCRsare then evolved from mixing day until round 10. In this way, we modelthe independent response in different lymph nodes against each strainthat occurs early on and the combined response in a typical lymph nodeagainst all strains that occurs after the lymph system has mixed.

6.8 References

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(2002). Immunization against a dominant tumor    antigen abrogates immunogenicity of the tumor. Cancer Immunity 2, 4.-   Martin-Fontecha, A., Sebastiani, S., Hopken, U. E., Uguccioni, M.,    Lipp, M., A, A. L., and Sallusto, F. (2003). Regulation of dendritic    cell migration to the draining lymph node: Impact on T lymphocyte    traffic and priming. J. Exp. Med. 198, 615-621.-   Mathew, A., Kurane, I., Rothman, A. L., Zeng, L. L., Brinton, M. A.,    and Ennis, F. A. (1996). Dominant recognition by human CD8+    cytotoxic T lymphocytes of dengue virus nonstructural proteins NS3    and NS1.2a. J. Clin. Invest. 98, 1684-1692.-   McMichael, A. J. and Rowland-Jones, S. L. (2001). Cellular immune    responses to HIV. Nature 410, 980-987.-   Mongkolsapaya, J., Dejnirattisai, W., Xu, X., Vasanawathana, S.,    Tangthawornchaikul, N., Chairunsri, A., Sawasdivom, S., Duangchinda,    T., Dong, T., Rowland-Jones, S., Yenchitsomanus, P., McMichael, A.,    Malasit, P., and Screaton, G. (2003). 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7. EQUIVALENTS

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

1. A method of modulating the immune response to one or more viruses ina subject in need thereof comprising administering to the subject aplurality of compositions in amounts effective to modulate said immuneresponse, each composition being administered to a different site of thesubject wherein each composition comprises at least one purifiedantigenic molecule, wherein at least one purified antigenic molecule ineach composition comprises a set of one or more epitopes of one or moreviral strains, and wherein the one or more purified antigenic moleculesof each composition comprise in aggregate a set of epitopes distinctfrom that of any other composition so administered, wherein the distancebetween each pair of sites is greater than the distance between any twoanatomically distinct lymph nodes or groups of lymph nodes nearest eachsite and wherein the distinct epitopes between the compositions differfrom each other by an epitopic variance defined as the sum of the numberof non-conservative amino acid changes and one half of the number ofconservative amino acid changes divided by the total number of aminoacids in the epitope, and wherein the epitopic variance between thedistinct epitopes between the compositions is between 0.05-0.5.
 2. Themethod of claim 1, wherein at least two compositions do not compriseepitopes of the same viral strain.
 3. The method of claim 1, wherein notwo compositions comprise the same epitopes.
 4. The method of claim 1,wherein each composition comprises at least one purified antigenicmolecule comprising one or more subdominant epitopes of one or moreviruses.
 5. The method of claim 1, wherein each composition comprises atleast one purified antigenic molecule comprising one or more dominantepitopes of one or more viruses.
 6. The method of claim 1, wherein eachantigenic molecule is a peptide which consists of an amino acid sequence25 amino acids or less.
 7. The method of claim 6, wherein the amino acidsequence of each said peptide consists of 12 amino acids or less.
 8. Themethod of claim 7, wherein the amino acid sequence of each said peptideconsists of 9 amino acids.
 9. The method of claim 6, wherein theepitopes are epitopes for different major histocompatibility complex(“MHC”) alleles.
 10. The method of claim 9, wherein the epitopes areoverlapping epitopes for different major histocompatibility complex(“MHC”) alleles.
 11. The method of claim 1, wherein the plurality ofcompositions is two to ten compositions.
 12. The method of claim 1,wherein the virus is selected from the group consisting of a lymphocyticchoriomeningitis virus, a hepatitis B virus, an Epstein Barr-virus, aninfluenza virus, and a human immunodeficiency virus.
 13. The method ofclaim 1, wherein the virus is a member of the Flaviviridae family ofviruses.
 14. The method of claim 13, wherein the flavivirus is selectedfrom the group consisting of dengue, Kunjin, Japanese encephalitits,West Nile, and yellow fever virus.
 15. The method of claim 14, whereinthe virus is a dengue virus.
 16. The method of claim 1, wherein theplurality of compositions is two to four compositions.
 17. The method ofclaim 16, wherein the plurality of compositions is two to threecompositions.
 18. A method of modulating the immune response to one ormore viruses in a subject in need thereof comprising administering tothe subject a plurality of compositions in amounts effective to modulatesaid immune response, each composition being administered to a differentsite of the subject wherein each composition comprises at least onepurified antigenic molecule, wherein at least one purified antigenicmolecule in each composition comprises a set of one or more epitopes ofone or more viral strains, and wherein the one or more purifiedantigenic molecules of each composition comprise in aggregate a set ofepitopes distinct from that of any other composition so administered,wherein the distance between each pair of sites is greater than thedistance between any two anatomically distinct lymph nodes or groups oflymph nodes nearest each site and wherein the distinct epitopes betweenthe compositions differ from each other by an epitopic variance definedas the sum of the number of non-conservative amino acid changes and onehalf of the number of conservative amino acid changes divided by thetotal number of amino acids in the epitope, and wherein the epitopicvariance between the distinct epitopes between the compositions isbetween 0.02-0.4.
 19. A kit comprising in separate containers at leasttwo compositions, each composition comprising at least one antigenicmolecule, wherein at least one antigenic molecule in each compositioncomprises one or more epitopes of the same infectious agent or strainthereof and wherein the one or more molecules of each compositioncomprise in aggregate a set of epitopes distinct from that of said othercomposition or compositions, wherein the distinct epitopes between thecompositions differ from each other by an epitopic variance defined asthe sum of the number of non-conservative amino acid changes and onehalf of the number of conservative amino acid changes divided by thetotal number of amino acids in the epitope, and wherein the epitopicvariance between the distinct epitopes between the compositions isbetween 0.05-0.5 and instructions for administering each composition toa separate site of a subject.
 20. The kit of claim 19, comprising inseparate containers at least four compositions.
 21. The kit of claim 19,comprising in separate containers at least three compositions.
 22. A kitcomprising in separate containers at least two compositions, eachcomposition comprising at least one antigenic molecule, wherein at leastone antigenic molecule in each composition comprises one or moreepitopes of the same infectious agent or strain thereof and wherein theone or more molecules of each composition comprise in aggregate a set ofepitopes distinct from that of said other composition or compositions,wherein the distinct epitopes between the compositions differ from eachother by an epitopic variance defined as the sum of the number ofnon-conservative amino acid changes and one half of the number ofconservative amino acid changes divided by the total number of aminoacids in the epitope, and wherein the epitopic variance between thedistinct epitopes between the compositions is between 0.02-0.4 andinstructions for administering each composition to a separate site of asubject.